Compositions and methods of nucleic acid-targeting nucleic acids

ABSTRACT

This disclosure provides for compositions and methods for the use of nucleic acid-targeting nucleic acids and complexes thereof.

CROSS-REFERENCE

This application is a continuation application of U.S. patentapplication Ser. No. 14/206,319, filed on Mar. 12, 2014, which claimsthe benefit of U.S. Provisional Application No. 61/818,386, filed May 1,2013 [Attorney Docket No. 44287-710.101], U.S. Provisional ApplicationNo. 61/902,723, filed Nov. 11, 2013 [Attorney Docket No. 44287-710.103],U.S. Provisional Application No. 61/818,382, filed May 1, 2013 [AttorneyDocket No. 44287-712.101], U.S. Provisional Application No. 61/859,661,filed Jul. 29, 2013 [Attorney Docket No. 44287-712.102], U.S.Provisional Application No. 61/858,767, filed Jul. 26, 2013 [AttorneyDocket No. 44287-713.102], U.S. Provisional Application No. 61/822,002,filed May 10, 2013 [Attorney Docket No. 44287-717.101], U.S. ProvisionalApplication No. 61/832,690, filed Jun. 7, 2013 [Attorney Docket No.44287-719.101], U.S. Provisional Application No. 61/906,211, filed Nov.19, 2013 [Attorney Docket No. 44287-719.102], U.S. ProvisionalApplication No. 61/900,311, filed Nov. 5, 2013 [Attorney Docket No.44287-719.103], U.S. Provisional Application No. 61/845,714, filed Jul.12, 2013 [Attorney Docket No. 44287-721.101], U.S. ProvisionalApplication No. 61/883,804, filed Sep. 27, 2013 [Attorney Docket No.44287-721.102], U.S. Provisional Application No. 61/781,598, filed Mar.14, 2013 [Attorney Docket No. 44287-722.101], U.S. ProvisionalApplication No. 61/899,712, filed Nov. 4, 2013 [Attorney Docket No.44287-727.101], U.S. Provisional Application No. 61/865,743, filed Aug.14, 2013 [Attorney Docket No. 44287-733.101], U.S. ProvisionalApplication No. 61/907,777, filed Nov. 22, 2013 [Attorney Docket No.44287-734.101], U.S. Provisional Application No. 61/903,232, filed Nov.12, 2013 [Attorney Docket No. 44287-751.101], U.S. ProvisionalApplication No. 61/906,335, filed Nov. 19, 2013 [Attorney Docket No.44287-752.101], U.S. Provisional Application No. 61/907,216, filed Nov.21, 2013 [Attorney Docket No. 44287-753.101], the entire contents ofwhich are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 10, 2014, isnamed 44287-722-201_SeqList_.txt and is 7,828,921 bytes in size.

BACKGROUND OF THE INVENTION

Genome engineering can refer to altering the genome by deleting,inserting, mutating, or substituting specific nucleic acid sequences.The altering can be gene or location specific. Genome engineering canuse nucleases to cut a nucleic acid thereby generating a site for thealteration. Engineering of non-genomic nucleic acid is alsocontemplated. A protein containing a nuclease domain can bind and cleavea target nucleic acid by forming a complex with a nucleic acid-targetingnucleic acid. In one example, the cleavage can introduce double strandedbreaks in the target nucleic acid. A nucleic acid can be repaired e.g.by endogenous non-homologous end joining (NHEJ) machinery. In a furtherexample, a piece of nucleic acid can be inserted. NModifications ofnucleic acid-targeting nucleic acids and site-directed polypeptides canintroduce new functions to be used for genome engineering.

SUMMARY OF THE INVENTION

In one aspect, the disclosure provides for an engineered nucleicacid-targeting nucleic acid comprising: a mutation in a P-domain of thenucleic acid-targeting nucleic acid. In some embodiments, the P-domainstarts downstream of a last paired nucleotide of a duplex between aCRISPR repeat and a tracrRNA sequence of the nucleic acid-targetingnucleic acid. In some embodiments, the engineered nucleic acid-targetingnucleic acid further comprises a linker sequence. In some embodiments,the linker sequence links the CRISPR repeat and the tracrRNA sequence.In some embodiments, the engineered nucleic acid-targeting nucleic acidis an isolated engineered nucleic acid-targeting nucleic acid. In someembodiments, the engineered nucleic acid-targeting nucleic acid is arecombinant engineered nucleic acid-targeting nucleic acid. In someembodiments, the engineered nucleic acid-targeting nucleic acid isadapted to hybridize to a target nucleic acid. In some embodiments, theP-domain comprises 2 adjacent nucleotides. In some embodiments, theP-domain comprises 3 adjacent nucleotides. In some embodiments, theP-domain comprises 4 adjacent nucleotides. In some embodiments, theP-domain comprises 5 adjacent nucleotides. In some embodiments, theP-domain comprises 6 or more adjacent nucleotides. In some embodiments,the P-domain starts 1 nucleotide downstream of the last pairednucleotide of the duplex. In some embodiments, the P-domain starts 2nucleotides downstream of the last paired nucleotide of the duplex. Insome embodiments, the P-domain starts 3 nucleotides downstream of thelast paired nucleotide of the duplex. In some embodiments, the P-domainstarts 4 nucleotides downstream of the last paired nucleotide of theduplex. In some embodiments, the P-domain starts 5 nucleotidesdownstream of the last paired nucleotide of the duplex. In someembodiments, the P-domain starts 6 or more nucleotides downstream of thelast paired nucleotide of the duplex. In some embodiments, the mutationcomprises one or more mutations. In some embodiments, the one or moremutations are adjacent to each other. In some embodiments, the one ormore mutations are separated from each other. In some embodiments, themutation is adapted to allow the engineered nucleic acid-targetingnucleic acid to hybridize to a different protospacer adjacent motif. Insome embodiments, the different protospacer adjacent motif comprises atleast 4 nucleotides. In some embodiments, the different protospaceradjacent motif comprises at least 5 nucleotides. In some embodiments,the different protospacer adjacent motif comprises at least 6nucleotides. In some embodiments, the different protospacer adjacentmotif comprises at least 7 or more nucleotides. In some embodiments, thedifferent protospacer adjacent motif comprises two non-adjacent regions.In some embodiments, the different protospacer adjacent motif comprisesthree non-adjacent regions. In some embodiments, the mutation is adaptedto allow the engineered nucleic acid-targeting nucleic acid to bind to atarget nucleic acid with a lower dissociation constant than anun-engineered nucleic acid-targeting nucleic acid. In some embodiments,the mutation is adapted to allow the engineered nucleic acid-targetingnucleic acid to bind to a target nucleic acid with greater specificitythan an un-engineered nucleic acid-targeting nucleic acid. In someembodiments, the mutation is adapted to reduce binding of the engineerednucleic acid-targeting nucleic acid to a non-specific sequence in atarget nucleic acid than an un-engineered nucleic acid-targeting nucleicacid. In some embodiments, the engineered nucleic acid-targeting nucleicacid further comprises two hairpins, wherein one of the two hairpinscomprises a duplex between a polynucleotide comprising at least 50%identity to a CRISPR RNA over 6 contiguous nucleotides, and apolynucleotide comprising at least 50% identity to a tracrRNA over 6contiguous nucleotides; and, wherein one of the two hairpins is 3′ ofthe first hairpin, wherein the second hairpin comprises an engineeredP-domain. In some embodiments, the second hairpin is adapted tode-duplex when the nucleic acid is in contact with a target nucleicacid. In some embodiments, the P-domain is adapted to: hybridize with afirst polynucleotide, wherein the first polynucleotide comprises aregion of the engineered nucleic acid-targeting nucleic acid, hybridizeto a second polynucleotide, wherein the second polynucleotide comprisesa target nucleic acid, and hybridize specifically to the first or secondpolynucleotide. In some embodiments, the first polynucleotide comprisesat least 50% identity to a tracrRNA over 6 contiguous nucleotides. Insome embodiments, the first polynucleotide is located downstream of aduplex between a polynucleotide comprising at least 50% identity to aCRISPR repeat over 6 contiguous nucleotides, and a polynucleotidecomprising at least 50% identity to a tracrRNA sequence over 6contiguous nucleotides. In some embodiments, the second polynucleotidecomprises a protospacer adjcent motif. In some embodiments, theengineered nucleic acid-targeting nucleic acid is adapted to bind to asite-directed polypeptide. In some embodiments, the mutation comprisesan insertion of one or more nucleotides into the P-domain. In someembodiments, the mutation comprises deletion one or more nucleotidesfrom the P-domain. In some embodiments, the mutation comprises mutationof one or more nucleotides. In some embodiments, the mutation isconfigured to allow the nucleic acid-targeting nucleic acid to hybridizeto a different protospacer adjacent motif. In some embodiments, thedifferent protospacer adjacent motif comprises a protospacer adjacentmotif selected from the group consisting of: 5′-NGGNG-3′,5′-NNAAAAW-3,5′-NNNNGATT-3,5′-GNNNCNNA-3 and 5′-NNNACA-3′, or anycombination thereof. In some embodiments, the mutation is configured toallow the engineered nucleic acid-targeting nucleic acid to bind with alower dissociation constant than an un-engineered nucleic acid-targetingnucleic acid. In some embodiments, the mutation is configured to allowthe engineered nucleic acid-targeting nucleic acid to bind with greaterspecificity than an un-engineered nucleic acid-targeting nucleic acid.In some embodiments, the mutation is configured to reduce binding of theengineered nucleic acid-targeting nucleic acid to a non-specificsequence in a target nucleic acid than an un-engineered nucleicacid-targeting nucleic acid.

In one aspect, the disclosure provides for a method for modifying atarget nucleic acid comprising contacting a target nucleic acid with anengineered nucleic acid-targeting nucleic acid comprising: a mutation ina P-domain of the nucleic acid-targeting nucleic acid, and modifying thetarget nucleic acid. In some embodiments, the method further comprisesinserting a donor polynucleotide into the target nucleic acid. In someembodiments, the modifying comprises cleaving the target nucleic acid.In some embodiments, the modifying comprises modifying transcription ofthe target nucleic acid.

In one aspect the disclosure provides for a vector comprising apolynucleotide sequence encoding an engineered nucleic acid-targetingnucleic acid comprising: a mutation in a P-domain of the nucleicacid-targeting nucleic acid.

In one aspect the disclosure provides for a kit comprising: anengineered nucleic acid-targeting nucleic acid comprising: a mutation ina P-domain of the nucleic acid-targeting nucleic acid; and a buffer. Insome embodiments, the kit further comprises a site-directed polypeptide.In some embodiments, the kit further comprises a donor polynucleotide.In some embodiments, the kit further comprises instructions for use.

In one aspect, the disclosure provides for an engineered nucleicacid-targeting nucleic acid comprising: a mutation in a bulge region ofa nucleic acid-targeting nucleic acid. In some embodiments, the bulge islocated within a duplex between a CRISPR repeat and a tracrRNA sequenceof the nucleic acid-targeting nucleic acid. In some embodiments, theengineered nucleic acid-targeting nucleic acid further comprises alinker sequence. In some embodiments, the linker sequence links theCRISPR repeat and the tracrRNA sequence. In some embodiments, theengineered nucleic acid-targeting nucleic acid is an isolated engineerednucleic acid-targeting nucleic acid. In some embodiments, the engineerednucleic acid-targeting nucleic acid is a recombinant engineered nucleicacid-targeting nucleic acid. In some embodiments, the bulge comprises atleast 1 unpaired nucleotide on the CRISPR repeat, and 1 unpairednucleotide on a the tracrRNA sequence. In some embodiments, the bulgecomprises at least 1 unpaired nucleotide on the CRISPR repeat, and atleast 2 unpaired nucleotides on a the tracrRNA sequence. In someembodiments, the bulge comprises at least 1 unpaired nucleotide on theCRISPR repeat, and at least 3 unpaired nucleotides on a the tracrRNAsequence. In some embodiments, the bulge comprises at least 1 unpairednucleotide on the CRISPR repeat, and at least 4 unpaired nucleotides ona the tracrRNA sequence. In some embodiments, the bulge comprises atleast one 1 unpaired nucleotide on the CRISPR repeat, and at least 5unpaired nucleotides on a the tracrRNA sequence. In some embodiments,the bulge comprises at least 2 unpaired nucleotide on the CRISPR repeat,and 1 unpaired nucleotide on a the tracrRNA sequence. In someembodiments, the bulge comprises at least 3 unpaired nucleotide on theCRISPR repeat, and at least 2 unpaired nucleotides on a the tracrRNAsequence. In some embodiments, the bulge comprises at least 4 unpairednucleotide on the CRISPR repeat, and at least 3 unpaired nucleotides ona the tracrRNA sequence. In some embodiments, the bulge comprises atleast 5 unpaired nucleotide on the CRISPR repeat, and at least 4unpaired nucleotides on the tracrRNA sequence. In some embodiments, thebulge comprises at least one nucleotide on the CRISPR repeat adapted toform a wobble pair with at least one nucleotide on the tracrRNAsequence. In some embodiments, the mutation comprises one or moremutations. In some embodiments, the one or more mutations are adjacentto each other. In some embodiments, the one or more mutations areseparated from each other. In some embodiments, the mutation is adaptedto allow the engineered nucleic acid-targeting nucleic acid to bind to adifferent site-directed polypeptide. In some embodiments, the differentsite-directed polypeptide is a homologue of Cas9. In some embodiments,the different site-directed polypeptide is a mutated version of Cas9. Insome embodiments, the different site-directed polypeptide comprises 10%amino acid sequence identity to Cas9 in a nuclease domain selected fromthe group consisting of: a RuvC nuclease domain, and a HNH nucleasedomain, or any combination thereof. In some embodiments, the mutation isadapted to allow the engineered nucleic acid-targeting nucleic acid tohybridize to a different protospacer adjacent motif. In someembodiments, the mutation is adapted to allow the engineered nucleicacid-targeting nucleic acid to bind to a site-directed polypeptide witha lower dissociation constant than an un-engineered nucleicacid-targeting nucleic acid. In some embodiments, the mutation isadapted to allow the engineered nucleic acid-targeting nucleic acid tobind to a site-directed polypeptide with greater specificity than anun-engineered nucleic acid-targeting nucleic acid. In some embodiments,the mutation is adapted to allow the engineered nucleic acid-targetingnucleic acid to direct a site-directed polypeptide to cleave a targetnucleic acid with greater specificity than an un-engineered nucleicacid-targeting nucleic acid. In some embodiments, the mutation isadapted to reduce binding of the engineered nucleic acid-targetingnucleic acid to a non-specific sequence in a target nucleic acid than anun-engineered nucleic acid-targeting nucleic acid. In some embodiments,the engineered nucleic acid-targeting nucleic acid is adapted tohybridize to a target nucleic acid. In some embodiments, the mutationcomprises insertion one or more nucleotides into the bulge. In someembodiments, the mutation comprises deletion of one or more nucleotidesfrom the bulge. In some embodiments, the mutation comprises mutation ofone or more nucleotides. In some embodiments, the mutation is configuredto allow the engineered nucleic acid-targeting nucleic acid to hybridizeto a different protospacer adjacent motif compared to an un-engineerednucleic acid-targeting nucleic acid. In some embodiments, the mutationis configured to allow the engineered nucleic acid-targeting nucleicacid to bind to a site-directed polypeptide with a lower dissociationconstant than an un-engineered nucleic acid-targeting nucleic acid. Insome embodiments, the mutation is configured to allow the engineerednucleic acid-targeting nucleic acid to bind to a site-directedpolypeptide with greater specificity than an un-engineered nucleicacid-targeting nucleic acid. In some embodiments, the mutation isconfigured to reduce binding of the engineered nucleic acid-targetingnucleic acid to a non-specific sequence in a target nucleic acid than anun-engineered nucleic acid-targeting nucleic acid.

In one aspect the disclosure provides for a method for modifying atarget nucleic acid comprising: contacting the target nucleic acid withan engineered nucleic acid-targeting nucleic acid comprising: a mutationin a bulge region of a nucleic acid-targeting nucleic acid; andmodifying the target nucleic acid. In some embodiments, the methodfurther comprises inserting a donor polynucleotide into the targetnucleic acid. In some embodiments, the modifying comprises cleaving thetarget nucleic acid. In some embodiments, the modifying comprisesmodifying transcription of the target nucleic acid.

In one aspect the disclosure provides for a vector comprising apolynucleotide sequence encoding an engineered nucleic acid-targetingnucleic acid comprising: a mutation in a bulge region of a nucleicacid-targeting nucleic acid; and modifying the target nucleic acid.

In one aspect the disclosure provides for a kit comprising: anengineered nucleic acid-targeting nucleic acid comprising: a mutation ina bulge region of a nucleic acid-targeting nucleic acid; and modifyingthe target nucleic acid; and a buffer. In some embodiments, the kitfurther comprises a site-directed polypeptide. In some embodiments, thekit further comprises a donor polynucleotide. In some embodiments, thekit further comprises instructions for use.

In one aspect the disclosure provides for a method for producing a donorpolynucleotide-tagged cell comprising: cleaving a target nucleic acid ina cell using a complex comprising a site-directed polypeptide and anucleic acid-targeting nucleic acid, inserting a donor polynucleotideinto a cleaved target nucleic acid, propagating the cell carrying thedonor polynucleotide, and determining an origin of thedonor-polynucleotide tagged cell. In some embodiments, the method isperformed in vivo. In some embodiments, the method is performed invitro. In some embodiments, the method is performed in situ. In someembodiments, the propagating produces a population of cells. In someembodiments, the propagating produces a cell line. In some embodiments,the method further comprises determining a nucleic acid sequence of anucleic acid in the cell. In some embodiments, the nucleic acid sequencedetermines an origin of the cell. In some embodiments, the determiningcomprises determining a genotype of the cell. In some embodiments, thepropagating comprises differentiating the cell. In some embodiments, thepropagating comprises de-differentiating the cell. In some embodiments,the propagating comprises differentiating the cell and thendedifferentiating the cell. In some embodiments, the propagatingcomprises passaging the cell. In some embodiments, the propagatingcomprises inducing the cell to divide. In some embodiments, thepropagating comprises inducing the cell to enter the cell cycle. In someembodiments, the propagating comprises the cell forming a metastasis. Insome embodiments, the propagating comprises differentiating apluripotent cell into a differentiated cell. In some embodiments, thecell is a differentiated cell. In some embodiments, the cell is ade-differentiated cell. In some embodiments, the cell is a stem cell. Insome embodiments, the cell is a pluripotent stem cell. In someembodiments, the cell is a eukaryotic cell line. In some embodiments,the cell is a primary cell line. In some embodiments, the cell is apatient-derived cell line. In some embodiments, the method furthercomprises transplanting the cell into an organism. In some embodiments,the organism is a human. In some embodiments, the organism is a mammal.In some embodiments, the organism is selected from the group consistingof: a human, a dog, a rat, a mouse, a chicken, a fish, a cat, a plant,and a primate. In some embodiments, the method further comprisesselecting the cell. In some embodiments, the donor polynucleotide isinserted into a target nucleic acid that is expressed in one cell state.In some embodiments, the donor polynucleotide is inserted into a targetnucleic acid that is expressed in a plurality of cell types. In someembodiments, the donor polynucleotide is inserted into a target nucleicacid that is expressed in a pluripotent state. In some embodiments, thedonor polynucleotide is inserted into a target nucleic acid that isexpressed in a differentiated state.

In one aspect the disclosure provides for a method for making a clonallyexpanded cell line comprising: introducing into a cell a complexcomprising: a site-directed polypeptide and a nucleic acid-targetingnucleic acid, contacting the complex to a target nucleic acid, cleavingthe target nucleic acid, wherein the cleaving is performed by thecomplex, thereby producing a cleaved target nucleic acid, inserting adonor polynucleotide into the cleaved target nucleic acid, propagatingthe cell, wherein the propagating produces the clonally expanded cellline. In some embodiments, the cell is selected from the groupconsisting of: HeLa cell, Chinese Hamster Ovary cell, 293-T cell, apheochromocytoma, a neuroblastomas fibroblast, a rhabdomyosarcoma, adorsal root ganglion cell, a NSO cell, CV-1 (ATCC CCL 70), COS-I (ATCCCRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C 1271 (ATCC CRL 1616),BS-C-I. (ATCC CCL 26), MRC-5 (ATCC CCL 171), L-cells, HEK-293 (ATCC CRL1573) and PC 12 (ATCC CRL-1721), HEK293T (ATCC CRL-11268), RBL (ATCCCRL-1378), SH-SY5Y (ATCC CRL-2266), MDCK (ATCC CCL-34), SJ-RH30 (ATCC2061), HepG2 (ATCC HB-8065), ND7/23 (ECACC 92090903), CHO (ECACC85050302), Vera (ATCC CCL 81), Caco-2 (ATCC HTB 37), K562 (ATCC CCL243), Jurkat (ATCC TIB-152), Per.Có, Huvec (ATCC Human Primary PCS100-010, Mouse CRL 2514, CRL 2515, CRL 2516), HuH-7D12 (ECACC 01042712),293 (ATCC CRL 10852), A549 (ATCC CCL 185), IMR-90 (ATCC CCL 186), MCF-7(ATC HTB-22), U-2 OS (ATCC HTB-96), and T84 (ATCC CCL 248), or anycombination thereof. In some embodiments, the cell is stem cell. In someembodiments, the cell is a differentiated cell. In some embodiments, thecell is a pluripotent cell.

In one aspect the disclosure provides for a method for multiplex celltype analysis comprising: cleaving at least one target nucleic acid intwo or more cells using a complex comprising a site-directed polypeptideand a nucleic acid-targeting nucleic acid, to create two cleaved targetnucleic acids, inserting a different a donor polynucleotide into each ofthe cleaved target nucleic acids, and analyzing the two or more cells.In some embodiments, the analyzing comprises simultaneously analyzingthe two or more cells. In some embodiments, the analyzing comprisesdetermining a sequence of the target nucleic acid. In some embodiments,the analyzing comprises comparing the two or more cells. In someembodiments, the analyzing comprises determining a genotype of the twoor more cells. In some embodiments, the cell is a differentiated cell.In some embodiments, the cell is a de-differentiated cell. In someembodiments, the cell is a stem cell. In some embodiments, the cell is apluripotent stem cell. In some embodiments, the cell is a eukaryoticcell line. In some embodiments, the cell is a primary cell line. In someembodiments, the cell is a patient-derived cell line. In someembodiments, a plurality of donor polynucleotides are inserted into aplurality of cleaved target nucleic acids in the cell.

In one aspect, the disclosure provides for a composition comprising: anengineered nucleic acid-targeting nucleic acid comprising a 3′hybridizing extension, and a donor polynucleotide, wherein the donorpolynucleotide is hybridized to the 3′ hybridizing extension. In someembodiments, the 3′ hybridizing extension is adapted to hybridize to atleast 5 nucleotides from the 3′ of the donor polynucleotide. In someembodiments, the 3′ hybridizing extension is adapted to hybridize to atleast 5 nucleotides from the 5′ of the donor polynucleotide. In someembodiments, the 3′ hybridizing extension is adapted to hybridize to atleast 5 adjacent nucleotides in the donor polynucleotide. In someembodiments, the 3′ hybridizing extension is adapted to hybridize to allof the donor polynucleotide. In some embodiments, the 3′ hybridizingextension comprises a reverse transcription template. In someembodiments, the reverse transcription template is adapted to be reversetranscribed by a reverse transcriptase. In some embodiments, thecomposition further comprises a reverse transcribed DNA polynucleotide.In some embodiments, the reverse transcribed DNA polynucleotide isadapted to hybridize to the reverse transcription template. In someembodiments, the donor polynucleotide is DNA. In some embodiments, the3′ hybridizing extension is RNA. In some embodiments, the engineerednucleic acid-targeting nucleic acid is an isolated engineered nucleicacid-targeting nucleic acid. In some embodiments, the engineered nucleicacid-targeting nucleic acid is a recombinant engineered nucleicacid-targeting nucleic acid.

In one aspect the disclosure provides for a method for introducing adonor polynucleotide into a target nucleic acid comprising: contactingthe target nucleic acid with a composition comprising: an engineerednucleic acid-targeting nucleic acid comprising a 3′ hybridizingextension, and a donor polynucleotide, wherein the donor polynucleotideis hybridized to the 3′ hybridizing extension. In some embodiments, themethod further comprises cleaving the target nucleic acid to produce acleaved target nucleic acid. In some embodiments, the cleaving isperformed by a site-directed polypeptide. In some embodiments, themethod further comprises inserting the donor polynucleotide into thecleaved target nucleic acid.

In one aspect, the disclosure provides for a composition comprising: aneffector protein, and a nucleic acid, wherein the nucleic acid comprisesat least 50% sequence identity to a crRNA over 6 contiguous nucleotides,at least 50% sequence identity to a tracrRNA over 6 contiguousnucleotides; and a non-native sequence, wherein the nucleic acid isadapted to bind to the effector protein. In some embodiments, thecomposition further comprises a polypeptide comprising at least 10%amino acid sequence identity to a nuclease domain of Cas9, wherein thenucleic acid binds to the polypeptide. In some embodiments, thepolypeptide comprises at least 60% amino acid sequence identity in anuclease domain to a nuclease domain of Cas9. In some embodiments, thepolypeptide is Cas9. In some embodiments, the nucleic acid furthercomprises a linker sequence, wherein the linker sequence links thesequence comprising at least 50% sequence identity to a crRNA over 6contiguous nucleotides and the sequence comprising at least 50% sequenceidentity to a tracrRNA over 6 contiguous nucleotides. In someembodiments, the non-native sequence is located at a position of thenucleic acid selected from the group consisting of: a 5′ end and a 3′end, or any combination thereof. In some embodiments, the nucleic acidcomprises two nucleic acid molecules. In some embodiments, the nucleicacid comprises a single continuous nucleic acid molecule. In someembodiments, the non-native sequence comprises a CRISPR RNA-bindingprotein binding sequence. In some embodiments, the non-native sequencecomprises a binding sequence selected from the group consisting of: aCas5 RNA-binding sequence, a Cas6 RNA-binding sequence, and a Csy4RNA-binding sequence, or any combination thereof. In some embodiments,the effector protein comprises a CRISPR RNA-binding protein. In someembodiments, the effector protein comprises at least 15% amino acidsequence identity to a protein selected from the group consisting of:Cas5, Cas6, and Csy4, or any combination thereof. In some embodiments,an RNA-binding domain of the effector protein comprises at least 15%amino acid sequence identity to an RNA-binding domain of a proteinselected from the group consisting of: Cas5, Cas6, and Csy4, or anycombination thereof. In some embodiments, the effector protein isselected from the group consisting of: Cas5, Cas6, and Csy4, or anycombination thereof. In some embodiments, the effector protein furthercomprises one or more non-native sequences. In some embodiments, thenon-native sequence confers an enzymatic activity to the effectorprotein. In some embodiments, the enzymatic activity is selected fromthe group consisting of: methyltransferase activity, demethylaseactivity, acetylation activity, deacetylation activity, ubiquitinationactivity, deubiquitination activity, deamination activity, dismutaseactivity, alkylation activity, depurination activity, oxidationactivity, pyrimidine dimer forming activity, transposase activity,recombinase activity, polymerase activity, ligase activity, helicaseactivity, photolyase activity or glycosylase activity, acetyltransferaseactivity, deacetylase activity, kinase activity, phosphatase activity,ubiquitin ligase activity, deubiquitinating activity, adenylationactivity, deadenylation activity, SUMOylating activity, deSUMOylatingactivity, ribosylation activity, deribosylation activity, myristoylationactivity, remodelling activity, protease activity, oxidoreductaseactivity, transferase activity, hydrolase activity, lyase activity,isomerase activity, synthase activity, synthetase activity, anddemyristoylation activity, or any combination thereof. In someembodiments, the nucleic acid is RNA. In some embodiments, the effectorprotein comprises a fusion protein comprising an RNA-binding protein anda DNA-binding protein. In some embodiments, the composition furthercomprises a donor polynucleotide. In some embodiments, the donorpolynucleotide is bound directly to the DNA binding protein, and whereinthe RNA binding protein is bound to the nucleic acid-targeting nucleicacid. In some embodiments, the 5′ end of the donor polynucleotide isbound to the DNA-binding protein. In some embodiments, the 3′ end of thedonor polynucleotide is bound to the DNA-binding protein. In someembodiments, at least 5 nucleotides of the donor polynucleotide bind tothe DNA-binding protein. In some embodiments, the nucleic acid is anisolated nucleic acid. In some embodiments, the nucleic acid is arecombinant nucleic acid.

In one aspect, the disclosure provides for a method for introducing adonor polynucleotide into a target nucleic acid comprising: contacting atarget nucleic acid with a complex comprising a site-directedpolypeptide and a composition comprising: an effector protein, and anucleic acid, wherein the nucleic acid comprises at least 50% sequenceidentity to a crRNA over 6 contiguous nucleotides, at least 50% sequenceidentity to a tracrRNA over 6 contiguous nucleotides; and a non-nativesequence, wherein the nucleic acid is adapted to bind to the effectorprotein. In some embodiments, the method further comprises cleaving thetarget nucleic acid. In some embodiments, the cleaving is performed bythe site-directed polypeptide. In some embodiments, the method furthercomprises inserting the donor polynucleotide into the target nucleicacid.

In one aspect the disclosure provides for a method for modulating atarget nucleic acid comprising: contacting a target nucleic acid withone or more complexes, each complex comprising a site-directedpolypeptide and a composition comprising: an effector protein, and anucleic acid, wherein the nucleic acid comprises at least 50% sequenceidentity to a crRNA over 6 contiguous nucleotides, at least 50% sequenceidentity to a tracrRNA over 6 contiguous nucleotides; and a non-nativesequence, wherein the nucleic acid is adapted to bind to the effectorprotein, and modulating the target nucleic acid. In some embodiments,the site-directed polypeptide comprises at least 10% amino acid sequenceidentity to a nuclease domain of Cas9. In some embodiments, themodulating is performed by the effector protein. In some embodiments,the modulating comprises an activity selected from the group consistingof: methyltransferase activity, demethylase activity, acetylationactivity, deacetylation activity, ubiquitination activity,deubiquitination activity, deamination activity, dismutase activity,alkylation activity, depurination activity, oxidation activity,pyrimidine dimer forming activity, transposase activity, recombinaseactivity, polymerase activity, ligase activity, helicase activity,photolyase activity or glycosylase activity, acetyltransferase activity,deacetylase activity, kinase activity, phosphatase activity, ubiquitinligase activity, deubiquitinating activity, adenylation activity,deadenylation activity, SUMOylating activity, deSUMOylating activity,ribosylation activity, deribosylation activity, myristoylation activity,remodelling activity, protease activity, oxidoreductase activity,transferase activity, hydrolase activity, lyase activity, isomeraseactivity, synthase activity, synthetase activity, and demyristoylationactivity, or any combination thereof. In some embodiments, the effectorprotein comprises one or more effector proteins.

In one aspect the disclosure provides for a method for detecting if twocomplexes are in proximity to one another comprising: contacting a firsttarget nucleic acid with a first complex, wherein the first complexcomprises a first site-directed polypeptide, a first modified nucleicacid-targeting nucleic acid, and a first effector protein, wherein theeffector protein is adapted to bind to the modified nucleicacid-targeting nucleic acid, and wherein the first effector proteincomprises a non-native sequence that comprises a first portion of asplit system, and contacting a second target nucleic acid with a secondcomplex, wherein the second complex comprises a second site-directedpolypeptide, a second modified nucleic acid-targeting nucleic acid, anda second effector protein, wherein the effector protein is adapted tobind to the modified nucleic acid-targeting nucleic acid, and whereinthe second effector protein comprises a non-native sequence thatcomprises a second portion of a split system. In some embodiments, thefirst target nucleic acid and the second target nucleic acid are on thesame polynucleotide polymer. In some embodiments, the split systemcomprises two or more protein fragments that individually are notactive, but, when formed into a complex, result in an active proteincomplex. In some embodiments, the method further comprises detecting aninteraction between the first portion and the second portion. In someembodiments, the detecting indicates the first and second complex are inproximity to one another. In some embodiments, the site-directedpolypeptide is adapted to be unable to cleave the target nucleic acid.In some embodiments, the detecting comprises determining the occurrenceof a genetic mobility event. In some embodiments, the genetic mobilityevent comprises a translocation. In some embodiments, prior to thegenetic mobility event the two portions of the split system do notinteract. In some embodiments, after the genetic mobility event the twoportions of the split system do interact. In some embodiments, thegenetic mobility event is a translocation between a BCR and an Abl gene.In some embodiments, the interaction activates the split system. In someembodiments, the interaction indicates the target nucleic acids bound bythe complexes are close together. In some embodiments, the split systemis selected from the group consisting of: split GFP system, a splitubiquitin system, a split transcription factor system, and a splitaffinity tag system, or any combination thereof. In some embodiments,the split system comprises a split GFP system. In some embodiments, thedetecting indicates a genotype. In some embodiments, the method furthercomprises: determining a course of treatment for a disease based on thegenotype. In some embodiments, the method further comprises treating thedisease. In some embodiments, the treating comprises administering adrug. In some embodiments, the treating comprises administering acomplex comprising a nucleic acid-targeting nucleic acid and asite-directed polypeptide, wherein the complex can modify a geneticelement involved in the disease. In some embodiments, the modifying isselected from the group consisting of: adding a nucleic acid sequence tothe genetic element, substituting a nucleic acid sequence in the geneticelement, and deleting a nucleic acid sequence from the genetic element,or any combination thereof. In some embodiments, the method furthercomprises: communicating the genotype from a caregiver to a patient. Insome embodiments, the communicating comprises communicating from astorage memory system to a remote computer. In some embodiments, thedetecting diagnoses a disease. In some embodiments, the method furthercomprises: communicating the diagnosis from a caregiver to a patient. Insome embodiments, the detecting indicates the presence of a singlenucleotide polymorphism (SNP). In some embodiments, the method furthercomprises: communicating the occurrence of a genetic mobility event froma caregiver to a patient. In some embodiments, the communicatingcomprises communicating from a storage memory system to a remotecomputer. In some embodiments, the site-directed polypeptide comprisesat least 20% amino acid sequence identity to Cas9. In some embodiments,the site-directed polypeptide comprises at least 60% amino acid sequenceidentity to Cas9. In some embodiments, the site-directed polypeptidecomprises at least 60% amino acid sequence identity in a nuclease domainto a nuclease domain of Cas9. In some embodiments, the site-directedpolypeptide is Cas9. In some embodiments, the modified nucleicacid-targeting nucleic acid comprises a non-native sequence. In someembodiments, the non-native sequence is located at a position of themodified nucleic acid-targeting nucleic acid selected from the groupconsisting of: a 5′ end, and a 3′ end, or any combination thereof. Insome embodiments, the modified nucleic acid-targeting nucleic acidcomprises two nucleic acid molecules. In some embodiments, the nucleicacid comprises a single continuous nucleic acid molecule comprising afirst portion comprising at least 50% identity to a CRISPR repeat over 6contiguous nucleotides and a second portion comprising at least 50%identity to a tracrRNA sequence over 6 contiguous nucleotides. In someembodiments, the first portion and the second portion are linked by alinker. In some embodiments, the non-native sequence comprises a CRISPRRNA-binding protein binding sequence. In some embodiments, thenon-native sequence comprises a binding sequence selected from the groupconsisting of: a Cas5 RNA-binding sequence, a Cas6 RNA-binding sequence,and a Csy4 RNA-binding sequence, or any combination thereof. In someembodiments, the modified nucleic acid-targeting nucleic acid is adaptedto bind to an effector protein. In some embodiments, the effectorprotein is a CRISPR RNA-binding protein. In some embodiments, theeffector protein comprises at least 15% amino acid sequence identity toa protein selected from the group consisting of: Cas5, Cas6, and Csy4,or any combination thereof. In some embodiments, a RNA-binding domain ofthe effector protein comprises at least 15% amino acid sequence identityto an RNA-binding domain of a protein selected from the group consistingof: Cas5, Cas6, and Csy4, or any combination thereof. In someembodiments, the effector protein is selected from the group consistingof: Cas5, Cas6, and Csy4, or any combination thereof. In someembodiments, the nucleic acid-targeting nucleic acid is RNA. In someembodiments, the target nucleic acid is DNA. In some embodiments, theinteraction comprises forming an affinity tag. In some embodiments, thedetecting comprises capturing the affinity tag. In some embodiments, themethod further comprises sequencing nucleic acid bound to the first andsecond complexes. In some embodiments, the method further comprisesfragmenting the nucleic acid prior to the capturing. In someembodiments, the interaction forms an activated system. In someembodiments, the method further comprises altering transcription of afirst target nucleic acid or a second target nucleic acid, wherein thealtering is performed by the activated system. In some embodiments, thesecond target nucleic acid is unattached to the first target nucleicacid. In some embodiments, the altering transcription of the secondtarget nucleic acid is performed in trans. In some embodiments, thealtering transcription of the first target nucleic acid is performed incis. In some embodiments, the first or second target nucleic acid isselected from the group consisting of: an endogenous nucleic acid, andan exogenous nucleic acid, or any combination thereof. In someembodiments, the altering comprises increasing transcription of thefirst or second target nucleic acids. In some embodiments, the first orsecond target nucleic acid comprises a polynucleotide encoding one ormore genes that cause cell death. In some embodiments, the first orsecond target nucleic acid comprises a polynucleotide encoding acell-lysis inducing peptide. In some embodiments, the first or secondtarget nucleic acid comprises a polynucleotide encoding an immune-cellrecruiting antigen. In some embodiments, the first or second targetnucleic acid comprises a polynucleotide encoding one or more genesinvolved in apoptosis. In some embodiments, the one or more genesinvolved in apoptosis comprises caspases. In some embodiments, the oneor more genes involved in apoptosis comprises cytokines. In someembodiments, the one or more genes involved in apoptosis are selectedfrom the group consisting of: tumor necrosis factor (TNF), TNF receptor1 (TNFR1), TNF receptor 2 (TNFR2), Fas receptor, FasL, caspase-8,caspase-10, caspase-3, caspase-9, caspase-3, caspase-6, caspase-7,Bcl-2, and apoptosis inducing factor (AlF), or any combination thereof.In some embodiments, the first or second target nucleic acid comprises apolynucleotide encoding one or more nucleic acid-targeting nucleicacids. In some embodiments, the one or more nucleic acid-targetingnucleic acids target a plurality of target nucleic acids. In someembodiments, the detecting comprises generating genetic data. In someembodiments, the method further comprises communicating the genetic datafrom a storage memory system to a remote computer. In some embodiments,the genetic data indicates a genotype. In some embodiments, the geneticdata indicates the occurrence of a genetic mobility event. In someembodiments, the genetic data indicates a spatial location of genes.

In one aspect, the disclosure provides for a kit comprising: asite-directed polypeptide, a modified nucleic acid-targeting nucleicacid, wherein the modified nucleic acid-targeting nucleic acid comprisesa non-native sequence, an effector protein that is adapted to bind tothe non-native sequence, and a buffer. In some embodiments, the kitfurther comprises instructions for use.

In one aspect the disclosure provides for a vector comprising apolynucleotide sequence encoding a modified nucleic acid-targetingnucleic acid, wherein the modified nucleic acid-targeting nucleic acidcomprises a non-native sequence. In some embodiments, the polynucleotidesequence is operably linked to a promoter. In some embodiments, thepromoter is an inducible promoter.

In one aspect the disclosure provides for a vector comprising: apolynucleotide sequence encoding: a modified nucleic acid-targetingnucleic acid, wherein the modified nucleic acid-targeting nucleic acidcomprises a sequence configured to bind to an effector protein, and asite-directed polypeptide. In some embodiments, the polynucleotidesequence is operably linked to a promoter. In some embodiments, thepromoter is an inducible promoter.

In one aspect, the disclosure provides for a vector comprising: apolynucleotide sequence encoding: a modified nucleic acid-targetingnucleic acid, wherein the modified nucleic acid-targeting nucleic acidcomprises a non-native sequence, a site-directed polypeptide, and aneffector protein. In some embodiments, the polynucleotide sequence isoperably linked to a promoter. In some embodiments, the promoter is aninducible promoter.

In one aspect the disclosure provides for a genetically modified cellcomprising a composition comprising: an effector protein, and a nucleicacid, wherein the nucleic acid comprises at least 50% sequence identityto a crRNA over 6 contiguous nucleotides, at least 50% sequence identityto a tracrRNA over 6 contiguous nucleotides; and a non-native sequence,wherein the nucleic acid is adapted to bind to the effector protein.

In one aspect the disclosure provides for a genetically modified cellcomprising a vector comprising a polynucleotide sequence encoding amodified nucleic acid-targeting nucleic acid, wherein the modifiednucleic acid-targeting nucleic acid comprises a non-native sequence.

In one aspect the disclosure provides for a genetically modified cellcomprising a vector comprising: a polynucleotide sequence encoding: amodified nucleic acid-targeting nucleic acid, wherein the modifiednucleic acid-targeting nucleic acid comprises a sequence configured tobind to an effector protein, and a site-directed polypeptide.

In one aspect the disclosure provides for a genetically modified cellcomprising a vector comprising: a polynucleotide sequence encoding: amodified nucleic acid-targeting nucleic acid, wherein the modifiednucleic acid-targeting nucleic acid comprises a non-native sequence, asite-directed polypeptide, and an effector protein.

In one aspect the disclosure provides for a kit comprising: a vectorcomprising a polynucleotide sequence encoding a modified nucleicacid-targeting nucleic acid, wherein the modified nucleic acid-targetingnucleic acid comprises a non-native sequence, and a buffer. In someembodiments, the kit further comprises instructions for use.

In one aspect the disclosure provides for a kit comprising: a vectorcomprising: a polynucleotide sequence encoding: a modified nucleicacid-targeting nucleic acid, wherein the modified nucleic acid-targetingnucleic acid comprises a sequence configured to bind to an effectorprotein, and a site-directed polypeptide, and a buffer. In someembodiments, the kit further comprises instructions for use.

In one aspect the disclosure provides for a kit comprising: a vectorcomprising: a polynucleotide sequence encoding: a modified nucleicacid-targeting nucleic acid, wherein the modified nucleic acid-targetingnucleic acid comprises a non-native sequence, a site-directedpolypeptide, and an effector protein, and a buffer. In some embodiments,the kit further comprises instructions for use.

In one aspect, the disclosure provides for a composition comprising: amultiplexed genetic targeting agent, wherein the multiplexed genetictargeting agent comprises one or more nucleic acid modules, wherein thenucleic acid module comprises a non-native sequence, and wherein thenucleic acid module is configured to bind to a polypeptide comprising atleast 10% amino acid sequence identity to a nuclease domain of Cas9 andwherein the nucleic acid module is configured to hybridize to a targetnucleic acid. In some embodiments, the nucleic acid module comprises afirst sequence comprising at least 50% sequence identity to a crRNA over6 contiguous nucleotides, and a second sequence comprising at least 50%sequence identity to a tracrRNA over 6 contiguous nucleotides. In someembodiments, the composition further comprises a linker sequence thatlinks the first and second sequences. In some embodiments, the one ormore nucleic acid modules hybridize to one or more target nucleic acids.In some embodiments, the one or more nucleic acid modules differ by atleast one nucleotide in a spacer region of the one or more nucleic acidmodules. In some embodiments, the one or more nucleic acid modules isRNA. In some embodiments, the multiplexed genetic targeting agent isRNA. In some embodiments, the non-native sequence comprises a ribozyme.In some embodiments, the non-native sequence comprises anendoribonuclease binding sequence. In some embodiments, theendoribonuclease binding sequence is located at a 5′ end of the nucleicacid module. In some embodiments, the endoribonuclease binding sequenceis located at a 3′ end of the nucleic acid module. In some embodiments,the endoribonuclease binding sequence is adapted to be bound by a CRISPRendoribonuclease. In some embodiments, the endoribonuclease bindingsequence is adapted to be bound by an endoribonuclease comprising a RAMPdomain. In some embodiments, the endoribonuclease binding sequence isadapted to be bound by an endoribonuclease selected from the groupconsisting of: a Cas5 superfamily member endoribonuclease, and a Cas6superfamily member endoribonuclease, or any combination thereof. In someembodiments, the endoribonuclease binding sequence is adapted to bebound by an endoribonuclease comprising at least 15% amino acid sequenceidentity to a protein selected from the group consisting of: Csy4, Cas5,and Cas6. In some embodiments, the endoribonuclease binding sequence isadapted to be bound by an endoribonuclease comprising at least 15% aminoacid sequence identity to a nuclease domain of a protein selected fromthe group consisting of: Csy4, Cas5, and Cas6. In some embodiments, theendoribonuclease binding sequence comprises a hairpin. In someembodiments, the hairpin comprises at least 4 consecutive nucleotides ina stem loop structure. In some embodiments, the endoribonuclease bindingsequence comprises at least 60% identity to a sequence selected from thegroup consisting of:

5′-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3′;5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′.5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′,5′-GUCGCCCCCCACGCGGGGGCGUGGAUUGAAAC-3′;5′-CCAGCCGCCUUCGGGCGGCUGUGUGUUGAAAC-3′;5′-GUCGCACUCUACAUGAGUGCGUGGAUUGAAAU-3′;5′-UGUCGCACCUUAUAUAGGUGCGUGGAUUGAAAU-3′; and5′-GUCGCGCCCCGCAUGGGGCGCGUGGAUUGAAA-3′,or any combination thereof. In some embodiments, the one or more nucleicacid modules are adapted to be bound by different endoribonucleases. Insome embodiments, the multiplexed genetic target agent is an isolatedmultiplexed genetic targeting agent. In some embodiments, themultiplexed genetic target agent is a recombinant multiplexed genetictarget agent.

In one aspect the disclosure provides for a vector comprising apolynucleotide sequence encoding a multiplexed genetic targeting agent,wherein the multiplexed genetic targeting agent comprises one or morenucleic acid modules, wherein the nucleic acid module comprises anon-native sequence, and wherein the nucleic acid module is configuredto bind to a polypeptide comprising at least 10% amino acid sequenceidentity to a nuclease domain of Cas9 and wherein the nucleic acidmodule is configured to hybridize to a target nucleic acid. In someembodiments, the polynucleotide sequence is operably linked to apromoter. In some embodiments, the promoter is an inducible promoter.

In one aspect, the disclosure provides for a genetically modified cellcomprising a multiplexed genetic targeting agent, wherein themultiplexed genetic targeting agent comprises one or more nucleic acidmodules, wherein the nucleic acid module comprises a non-nativesequence, and wherein the nucleic acid module is configured to bind to apolypeptide comprising at least 10% amino acid sequence identity to anuclease domain of Cas9 and wherein the nucleic acid module isconfigured to hybridize to a target nucleic acid.

In one aspect the disclosure provides for a genetically modified cellcomprising a vector comprising a polynucleotide sequence encoding amultiplexed genetic targeting agent, wherein the multiplexed genetictargeting agent comprises one or more nucleic acid modules, wherein thenucleic acid module comprises a non-native sequence, and wherein thenucleic acid module is configured to bind to a polypeptide comprising atleast 10% amino acid sequence identity to a nuclease domain of Cas9 andwherein the nucleic acid module is configured to hybridize to a targetnucleic acid.

In one aspect the disclosure provides for a kit comprising a multiplexedgenetic targeting agent, wherein the multiplexed genetic targeting agentcomprises one or more nucleic acid modules, wherein the nucleic acidmodule comprises a non-native sequence, and wherein the nucleic acidmodule is configured to bind to a polypeptide comprising at least 10%amino acid sequence identity to a nuclease domain of Cas9 and whereinthe nucleic acid module is configured to hybridize to a target nucleicacid, and a buffer. In some embodiments, the kit further comprisesinstructions for use.

In one aspect the disclosure provides for a kit comprising: a vectorcomprising a polynucleotide sequence encoding a multiplexed genetictargeting agent, wherein the multiplexed genetic targeting agentcomprises one or more nucleic acid modules, wherein the nucleic acidmodule comprises a non-native sequence, and wherein the nucleic acidmodule is configured to bind to a polypeptide comprising at least 10%amino acid sequence identity to a nuclease domain of Cas9 and whereinthe nucleic acid module is configured to hybridize to a target nucleicacid, and a buffer. In some embodiments, the kit further comprisesinstructions for use.

In one aspect the disclosure provides for a method for generating anucleic acid, wherein the nucleic acid binds to a polypeptide comprisingat least 10% amino acid sequence identity to a nuclease domain of Cas9and hybridizes to a target nucleic acid comprising: introducing the amultiplexed genetic targeting agent, wherein the multiplexed genetictargeting agent comprises one or more nucleic acid modules, wherein thenucleic acid module comprises a non-native sequence, and wherein thenucleic acid module is configured to bind to a polypeptide comprising atleast 10% amino acid sequence identity to a nuclease domain of Cas9 andwherein the nucleic acid module is configured to hybridize to a targetnucleic acidinto a host cell, processing the multiplexed genetictargeting agent into the one or more nucleic acid modules, andcontacting the processed one or more nucleic acid modules to one or moretarget nucleic acids in the cell. In some embodiments, the methodfurther comprises cleaving the target nucleic acid. In some embodiments,the method further comprises modifying the target nucleic acid. In someembodiments, the modifying comprises altering transcription of thetarget nucleic acid. In some embodiments, the modifying comprisesinserting a donor polynucleotide into the target nucleic acid.

In one aspect the disclosure provides for a modified site-directedpolypeptide comprising: a first nuclease domain, a second nucleasedomain, and an inserted nuclease domain. In some embodiments, thesite-directed polypeptide comprises at least 15% identity to a nucleasedomain of Cas9. In some embodiments, the first nuclease domain comprisesa nuclease domain selected from the group consisting of: a HNH domain,and a RuvC domain, or any combination thereof. In some embodiments, thesecond nuclease domain comprises a nuclease domain selected from thegroup consisting of: a HNH domain, and a RuvC domain, or any combinationthereof. In some embodiments, the inserted nuclease domain comprises aHNH domain. In some embodiments, the inserted nuclease domain comprisesa RuvC domain. In some embodiments, the inserted nuclease domain isN-terminal to the first nuclease domain. In some embodiments, theinserted nuclease domain is N-terminal to the second nuclease domain. Insome embodiments, the inserted nuclease domain is C-terminal to thefirst nuclease domain. In some embodiments, the inserted nuclease domainis C-terminal to the second nuclease domain. In some embodiments, theinserted nuclease domain is in tandem to the first nuclease domain. Insome embodiments, the inserted nuclease domain is in tandem to thesecond nuclease domain. In some embodiments, the inserted nucleasedomain is adapted to cleave a target nucleic acid at a site differentthan the first or second nuclease domains. In some embodiments, theinserted nuclease domain is adapted to cleave an RNA in a DNA-RNAhybrid. In some embodiments, the inserted nuclease domain is adapted tocleave a DNA in a DNA-RNA hybrid. In some embodiments, the insertednuclease domain is adapted to increase specificity of binding of themodified site-directed polypeptide to a target nucleic acid. In someembodiments, the inserted nuclease domain is adapted to increasestrength of binding of the modified site-directed polypeptide to atarget nucleic acid.

In one aspect the disclosure provides for a vector comprising apolynucleotide sequence encoding a modified site-directed polypeptidecomprising: a first nuclease domain, a second nuclease domain, and aninserted nuclease domain.

In one aspect the disclosure provides for a kit comprising: a modifiedsite-directed polypeptide comprising: a first nuclease domain, a secondnuclease domain, and an inserted nuclease domain, and a buffer. In someembodiments, the kit further comprises instructions for use.

In one aspect the disclosure provides for a composition comprising: amodified site-directed polypeptide, wherein the polypeptide is modifiedsuch that it is adapted to target a second protospacer adjacent motifcompared to a wild-type site-directed polypeptide. In some embodiments,the site-directed polypeptide is modified by a modification selectedfrom the group consisting of: an amino acid addition, an amino acidsubstitution, an amino acid replacement, and an amino acid deletion, orany combination thereof. In some embodiments, the modified site-directedpolypeptide comprises a non-native sequence. In some embodiments, themodified site-directed polypeptide is adapted to target the secondprotospacer adjacent motif with greater specificity than the wild-typesite-directed polypeptide. In some embodiments, the modifiedsite-directed polypeptide is adapted to target the second protospaceradjacent motif with a lower dissociation constant compared to thewild-type site-directed polypeptide. In some embodiments, the modifiedsite-directed polypeptide is adapted to target the second protospaceradjacent motif with a higher dissociation constant compared to thewild-type site-directed polypeptide. In some embodiments, the secondprotospacer adjacent motif comprises a protospacer adjacent motifselected from the group consisting of: 5′-NGGNG-3′,5′-NNAAAAW-3,5′-NNNNGATT-3,5′-GNNNCNNA-3′, and 5′-NNNACA-3′, or anycombination thereof.

In one aspect the disclosure provides for a vector comprising apolynucleotide sequence encoding a modified site-directed polypeptide,wherein the polypeptide is modified such that it is adapted to target asecond protospacer adjacent motif compared to a wild-type site-directedpolypeptide.

In one aspect the disclosure provides for a kit comprising: a modifiedsite-directed polypeptide, wherein the polypeptide is modified such thatit is adapted to target a second protospacer adjacent motif compared toa wild-type site-directed polypeptide, and a buffer. In someembodiments, the kit further comprises instructions for use.

In one aspect the disclosure provides for a composition comprising: amodified site-directed polypeptide, wherein the polypeptide is modifiedsuch that it is adapted to target a second nucleic acid-targetingnucleic acid compared to a wild-type site-directed polypeptide. In someembodiments, the site-directed polypeptide is modified by a modificationselected from the group consisting of: an amino acid addition, an aminoacid substitution, an amino acid replacement, and an amino aciddeletion, or any combination thereof. In some embodiments, the modifiedsite-directed polypeptide comprises a non-native sequence. In someembodiments, the modified site-directed polypeptide is adapted to targetthe second nucleic acid-targeting nucleic acid with greater specificitythan the wild-type site-directed polypeptide. In some embodiments, themodified site-directed polypeptide is adapted to target the secondnucleic acid-targeting nucleic acid with a lower dissociation constantcompared to the wild-type site-directed polypeptide. In someembodiments, the modified site-directed polypeptide is adapted to targetthe second nucleic acid-targeting nucleic acid with a higherdissociation constant compared to the wild-type site-directedpolypeptide. In some embodiments, the site-directed polypeptide targetsa tracrRNA portion of the second nucleic acid target nucleic acid.

In one aspect the disclosure provides for a vector comprising apolynucleotide sequence encoding a modified site-directed polypeptide,wherein the polypeptide is modified such that it is adapted to target asecond nucleic acid-targeting nucleic acid compared to a wild-typesite-directed polypeptide.

In one aspect the disclosure provides for a kit comprising: a modifiedsite-directed polypeptide, wherein the polypeptide is modified such thatit is adapted to target a second nucleic acid-targeting nucleic acidcompared to a wild-type site-directed polypeptide, and a buffer. In someembodiments, the kit further comprises instructions for use.

In one aspect the disclosure provides for a composition comprising: amodified site-directed polypeptide comprising a modification in a bridgehelix as compared to SEQ ID: 8. In some embodiments, the composition isconfigured to cleave a target nucleic acid.

In one aspect the disclosure provides for a composition comprising: amodified site-directed polypeptide comprising a modification in a highlybasic patch as compared to SEQ ID: 8. In some embodiments, thecomposition is configured to cleave a target nucleic acid.

In one aspect the disclosure provides for a composition comprising: amodified site-directed polypeptide comprising a modification in apolymerase-like domain as compared to SEQ ID: 8. In some embodiments,the composition is configured to cleave a target nucleic acid.

In one aspect the disclosure provides for a composition comprising: amodified site-directed polypeptide comprising a modification in a bridgehelix, highly basic patch, nuclease domain, and polymerase domain ascompared to SEQ ID: 8, or any combination thereof.

In one aspect the disclosure provides for a vector comprising apolynucleotide sequence encoding a modified site-directed polypeptidecomprising a modification in a bridge helix, highly basic patch,nuclease domain, and polymerase domain as compared to SEQ ID: 8, or anycombination thereof.

In one aspect the disclosure provides for a kit comprising: a modifiedsite-directed polypeptide comprising a modification in a bridge helix,highly basic patch, nuclease domain, and polymerase domain as comparedto SEQ ID: 8, or any combination thereof, and a buffer. In someembodiments, the kit further comprises instructions for use. In someembodiments, the kit further comprises a nucleic acid-targeting nucleicacid.

In one aspect the disclosure provides for a genetically modified cell amodified site-directed polypeptide comprising a modification in a bridgehelix, highly basic patch, nuclease domain, and polymerase domain ascompared to SEQ ID: 8, or any combination thereof.

In one aspect the disclosure provides for a method for genomeengineering comprising: contacting a target nucleic acid with a complex,wherein the complex comprises a modified site-directed polypeptidecomprising a modification in a bridge helix, highly basic patch,nuclease domain, and polymerase domain as compared to SEQ ID: 8, or anycombination thereoand a nucleic acid-targeting nucleic acid, andmodifying the target nucleic acid. In some embodiments, the contactingcomprises contacting the complex to a protospacer adjacent motif in thetarget nucleic acid. In some embodiments, the contacting comprisescontacting the complex to a longer target nucleic acid sequence comparedto an unmodified site-directed polypeptide. In some embodiments, themodifying comprises cleaving the target nucleic acid. In someembodiments, the target nucleic acid comprises RNA. In some embodiments,the target nucleic acid comprises DNA. In some embodiments, themodifying comprises cleaving the RNA strand of a hybridized RNA and DNA.In some embodiments, the modifying comprises cleaving the DNA strand ofa hybridized RNA and DNA. In some embodiments, the modifying comprisesinserting into the target nucleic acid a donor polynucleotide, a portionof a donor polynucleotide, a copy of a donor polynucleotide, or aportion of a copy of a donor polynucleotide, or any combination thereof.In some embodiments, the modifying comprises modifying transcriptionalactivity of the target nucleic acid. In some embodiments, the modifyingcomprises a deleting of one or more nucleotides of the target nucleicacid.

In one aspect the disclosure provides for a composition comprising: amodified site-directed polypeptide comprising a modified nuclease domainas compared to SEQ ID: 8. In some embodiments, the composition isconfigured to cleave a target nucleic acid. In some embodiments, themodified nuclease domain comprises a RuvC domain nuclease domain. Insome embodiments, the modified nuclease domain comprises an HNH nucleasedomain. In some embodiments, the modified nuclease domain comprisesduplication of an HNH nuclease domain. In some embodiments, the modifiednuclease domain is adapted to increase specificity of the amino acidsequence for a target nucleic acid compared to an unmodifiedsite-directed polypeptide. In some embodiments, the modified nucleasedomain is adapted to increase specificity of the amino acid sequence fora nucleic acid-targeting nucleic acid compared to an unmodifiedsite-directed polypeptide. In some embodiments, the modified nucleasedomain comprises a modification selected from the group consisting of:an amino acid addition, an amino acid substitution, an amino acidreplacement, and an amino acid deletion, or any combination thereof. Insome embodiments, the modified nuclease domain comprises an insertednon-native sequence. In some embodiments, the non-native sequenceconfers an enzymatic activity to the modified site-directed polypeptide.In some embodiments, the enzymatic activity is selected from the groupconsisting of: nuclease activity, methylase activity, acetylaseactivity, demethylase activity, deamination activity, dismutaseactivity, alkylation activity, depurination activity, oxidationactivity, pyrimidine dimer forming activity, integrase activity,transposase activity, recombinase activity, polymerase activity, ligaseactivity, helicase activity, photolyase activity or glycosylaseactivity, acetyltransferase activity, deacetylase activity, kinaseactivity, phosphatase activity, ubiquitin ligase activity,deubiquitinating activity, adenylation activity, deadenylation activity,SUMOylating activity, deSUMOylating activity, ribosylation activity,deribosylation activity, myristoylation activity, remodelling activity,protease activity, oxidoreductase activity, transferase activity,hydrolase activity, lyase activity, isomerase activity, synthaseactivity, synthetase activity, and demyristoylation activity, or anycombination thereof. In some embodiments, the enzymatic activity isadapted to modulate transcription of a target nucleic acid. In someembodiments, the modified nuclease domain is adapted to allow binding ofthe amino acid sequence to a protospacer adjacent motif sequence that isdifferent from a protospacer adjacent motif sequence to which anunmodified site-directed polypeptide is adapted to bind. In someembodiments, the modified nuclease domain is adapted to allow binding ofthe amino acid sequence to a nucleic acid-targeting nucleic acid that isdifferent from a nucleic acid-targeting nucleic acid to which anunmodified site-directed polypeptide is adapted to bind. In someembodiments, the modified site-directed polypeptide is adapted to bindto a longer target nucleic acid sequence than an unmodifiedsite-directed polypeptide. In some embodiments, the modifiedsite-directed polypeptide is adapted to cleave double-stranded DNA. Insome embodiments, the modified site-directed polypeptide is adapted tocleave the RNA strand of a hybridized RNA and DNA. In some embodiments,the modified site-directed polypeptide is adapted to cleave the DNAstrand of a hybridized RNA and DNA. In some embodiments, the compositionfurther comprises a modified nucleic acid-targeting nucleic acid,wherein the modification of the site-directed polypeptide is adapted toenable the site-directed polypeptide to bind to the modified nucleicacid-targeting nucleic acid. In some embodiments, the modified nucleicacid-targeting nucleic acid and the modified site-directed polypeptidecomprise compensatory mutations.

In one aspect the disclosure provides for a method for enriching atarget nucleic acid for sequencing comprising: contacting a targetnucleic acid with a complex comprising a nucleic acid-targeting nucleicacid and a site-directed polypeptide, enriching the target nucleic acidusing the complex, and determining a sequence of the target nucleicacid. In some embodiments, the method does not comprise an amplificationstep. In some embodiments, the method further comprises analyzing thesequence of the target nucleic acid. In some embodiments, the methodfurther comprises fragmenting the target nucleic acid prior to theenriching. In some embodiments, the nucleic acid-targeting nucleic acidcomprises RNA. In some embodiments, the method the nucleicacid-targeting nucleic acid comprises two RNA molecules. In someembodiments, the method a portion of each of the two RNA moleculeshybridize together. In some embodiments, the method one of the two RNAmolecules comprises a CRISPR repeat sequence. In some embodiments, theCRISPR repeat sequence is homologous to a crRNA over 6 contiguousnucleotides. In some embodiments, the CRISPR repeat sequence comprisesat least 60% identity to a crRNA over 6 contiguous nucleotides. In someembodiments, the one of the two RNA molecules comprises a tracrRNAsequence. In some embodiments, the tracRNA sequence is homologous to atracrRNA over 6 contiguous nucleotides. In some embodiments, the tracRNAsequence comprises at least 60% identity to a tracrRNA over 6 contiguousnucleotides. In some embodiments, the nucleic acid-targeting nucleicacid is a double guide nucleic acid. In some embodiments, the nucleicacid-targeting nucleic acid comprises one continuous RNA moleculewherein the continuous RNA molecule further comprises two domains and alinker. In some embodiments, a portion of each of the two domains of thecontinuous RNA molecule hybridize together. In some embodiments, thecontinuous RNA molecule comprises a CRISPR repeat sequence. In someembodiments, the CRISPR repeat sequence is homologous to a crRNA over 6contiguous nucleotides. In some embodiments, the CRISPR repeat sequencecomprises at least 60% identity to a crRNA over 6 contiguousnucleotides. In some embodiments, the continuous RNA molecule comprisesa tracrRNA sequence. In some embodiments, the tracRNA sequence ishomologous to a tracrRNA over 6 contiguous nucleotides. In someembodiments, the tracRNA sequence comprises at least 60% identity to atracrRNA over 6 contiguous nucleotides. In some embodiments, the nucleicacid-targeting nucleic acid is a single guide nucleic acid. In someembodiments, the contacting comprises hybridizing a portion of thenucleic acid-targeting nucleic acid with a portion of the target nucleicacid. In some embodiments, the nucleic acid-targeting nucleic acidhybridizes with the target nucleic acid over a region comprising 6-20nucleotides. In some embodiments, the site-directed polypeptidecomprises Cas9. In some embodiments, the site-directed polypeptidecomprises at least 20% homology to a nuclease domain of Cas9. In someembodiments, the site-directed polypeptide comprises at least 60%homology to Cas9. In some embodiments, the site-directed polypeptidecomprises an engineered nuclease domain wherein the nuclease domaincomprises reduced nuclease activity compared to a site-directedpolypeptide that comprises an unengineered nuclease domain. In someembodiments, the site-directed polypeptide introduces a single-strandbreak in the target nucleic acid. In some embodiments, the engineerednuclease domain comprises mutation of a conserved aspartic acid. In someembodiments, the engineered nuclease domain comprises a D10A mutation.In some embodiments, the engineered nuclease domain comprises mutationof a conserved histidine. In some embodiments, the engineered nucleasedomain comprises a H840A mutation. In some embodiments, thesite-directed polypeptide comprises an affinity tag. In someembodiments, the affinity tag is located at the N-terminus of thesite-directed polypeptide, the C-terminus of the site-directedpolypeptide, a surface-accessible region, or any combination thereof. Insome embodiments, the affinity tag is selected from a group comprising:biotin, FLAG, His6×, His9×, and a fluorescent protein, or anycombination thereof. In some embodiments, the nucleic acid-targetingnucleic acid comprises a nucleic acid affinity tag. In some embodiments,the nucleic acid affinity tag is located at the 5′ end of the nucleicacid-targeting nucleic acid, the 3′ end of the nucleic acid-targetingnucleic acid, a surface-accessible region, or any combination thereof.In some embodiments, the nucleic acid affinity tag is selected from thegroup comprising a small molecule, fluorescent label, a radioactivelabel, or any combination thereof. In some embodiments, the nucleic acidaffinity tag comprises a sequence that is configured to bind to Csy4,Cas5, Cas6, or any combination thereof. In some embodiments, the nucleicacid affinity tag comprises 50% identity to5′-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3′. In some embodiments, the methodfurther comprises diagnosing a disease and making a patient-specifictreatment decision, or any combination thereof. In some embodiments, thedetermining comprises determining a genotype. In some embodiments, themethod further comprises communicating the sequence from a storagememory system to a remote computer. In some embodiments, the enrichingcomprises contacting an affinity tag of the complex with a captureagent. In some embodiments, the capture agent comprises an antibody. Insome embodiments, the capture agent comprises a solid support. In someembodiments, the capture agent is selected from the group comprising:Csy4, Cas5, and Cas6. In some embodiments, the the capture agentcomprises reduced enzymatic activity in the absence of imidazole. Insome embodiments, the capture agent comprises an activatable enzymaticdomain, wherein the activatable enzymatic domain is activated by comingin contact with imidazole. In some embodiments, the capture agent is aCas6 family member. In some embodiments, the capture agent comprises anaffinity tag. In some embodiments, the capture agent comprises aconditionally enzymatically inactive endoribonuclease comprising amutation in a nuclease domain. In some embodiments, the mutation of aconserved histidine. In some embodiments, the mutation comprises a H29Amutation. In some embodiments, the target nucleic acid is bound to thecomplex. In some embodiments, the target nucleic acid is an excisednucleic acid that is not bound to the complex. In some embodiments, aplurality of complexes are contacted to a plurality of target nucleicacids. In some embodiments, the plurality of target nucleic acids differby at least one nucleotide. In some embodiments, the plurality ofcomplexes comprise a plurality of nucleic acid-targeting nucleic acidsthat differ by at least one nucleotide.

In one aspect the disclosure provides for a method for excising anucleic acid comprising: contacting a target nucleic acid with two ormore complexes, wherein each complex comprises a site-directedpolypeptide and a nucleic acid-targeting nucleic acid, and cleaving thetarget nucleic acid, wherein the cleaving produces an excised targetnucleic acid. In some embodiments, the cleaving is performed by anuclease domain of the site-directed polypeptide. In some embodiments,the method does not comprise amplification. In some embodiments, themethod further comprises enriching the excised target nucleic acid. Insome embodiments, the method further comprises sequencing the excisedtarget nucleic acid. In some embodiments, the nucleic acid-targetingnucleic acid is RNA. In some embodiments, the nucleic acid-targetingnucleic acid comprises two RNA molecules. In some embodiments, a portionof each of the two RNA molecules hybridize together. In someembodiments, one of the two RNA molecules comprises a CRISPR repeatsequence. In some embodiments, the CRISPR repeat sequence comprises asequence that is homologous to a crRNA over 6 contiguous nucleotides. Insome embodiments, the CRISPR repeat sequence comprises a sequence thathas at least 60% identity to a crRNA over 6 contiguous nucleotides. Insome embodiments, one of the two RNA molecules comprises a tracrRNAsequence. In some embodiments, the tracRNA sequence is homologous to acrRNA over 6 contiguous nucleotides. In some embodiments, the tracRNAsequence comprises at least 60% identity to a crRNA over 6 contiguousnucleotides. In some embodiments, the nucleic acid-targeting nucleicacid is a double guide nucleic acid. In some embodiments, the nucleicacid-targeting nucleic acid comprises one continuous RNA moleculewherein the continuous RNA molecule further comprises two domains and alinker. In some embodiments, a portion of each of the two domains of thecontinuous RNA molecule hybridize together. In some embodiments, thecontinuous RNA molecule comprises a CRISPR repeat sequence. In someembodiments, the CRISPR repeat sequence is homologous to a crRNA over 6contiguous nucleotides. In some embodiments, the CRISPR repeat sequencecomprises at least 60% identity to a crRNA over 6 contiguousnucleotides. In some embodiments, the continuous RNA molecule comprisesa tracrRNA sequence. In some embodiments, the tracRNA sequence ishomologous to a crRNA over 6 contiguous nucleotides. In someembodiments, the tracRNA sequence comprises at least 60% identity to acrRNA over 6 contiguous nucleotides. In some embodiments, the nucleicacid-targeting nucleic acid is a single guide nucleic acid. In someembodiments, the nucleic acid-targeting nucleic acid hybridizes with atarget nucleic acid. In some embodiments, the nucleic acid-targetingnucleic acid hybridizes with a target nucleic acid over a region,wherein the region comprises at least 6 nucleotides and at most 20nucleotides. In some embodiments, the site-directed polypeptide is Cas9.In some embodiments, the site-directed polypeptide comprises apolypeptide comprising at least 20% homology to a nuclease domain ofCas9. In some embodiments, the site-directed polypeptide comprises apolypeptide comprising at least 60% homology to Cas9. In someembodiments, the site-directed polypeptide comprises an affinity tag. Insome embodiments, the affinity tag is located at the N-terminus of thesite-directed polypeptide, the C-terminus of the site-directedpolypeptide, a surface-accessible region, or any combination thereof. Insome embodiments, the affinity tag is selected from a group comprising:biotin, FLAG, His6×, His9×, and a fluorescent protein, or anycombination thereof. In some embodiments, the nucleic acid-targetingnucleic acid comprises a nucleic acid affinity tag. In some embodiments,the nucleic acid affinity tag is located at the 5′ end of the nucleicacid-targeting nucleic acid, the 3′ end of the nucleic acid-targetingnucleic acid, a surface-accessible region, or any combination thereof.In some embodiments, the nucleic acid affinity tag is selected from thegroup comprising a small molecule, fluorescent label, a radioactivelabel, or any combination thereof. In some embodiments, the nucleic acidaffinity tag is a sequence that can bind to Csy4, Cas5, Cas6, or anycombination thereof. In some embodiments, the nucleic acid affinity tagcomprises 50% identity to GUUCACUGCCGUAUAGGCAGCUAAGAAA. In someembodiments, the target nucleic acid is an excised nucleic acid that isnot bound to the two or more complexes. In some embodiments, the two ormore complexes are contacted to a plurality of target nucleic acids. Insome embodiments, the plurality of target nucleic acids differ by atleast one nucleotide. In some embodiments, the two or more complexescomprise nucleic acid-targeting nucleic acids that differ by at leastone nucleotide.

In one aspect the disclosure provides for a method for generating alibrary of target nucleic acids comprising: contacting a plurality oftarget nucleic acids with a complex comprising a site-directedpolypeptide and a nucleic acid-targeting nucleic acid, cleaving theplurality of target nucleic acids, and purifying the plurality of targetnucleic acids to create the library of target nucleic acids. In someembodiments, the method further comprises screening the library oftarget nucleic acids.

In one aspect the disclosure provides for a composition comprising: afirst complex comprising: a first site-directed polypeptide and a firstnucleic acid-targeting nucleic acid, a second complex comprising: asecond site-directed polypeptide and a second nucleic acid-targetingnucleic acid, wherein, the first and second nucleic acid-targetingnucleic acids are different. In some embodiments, the compositionfurther comprises a target nucleic acid, which is bound by the first orthe second complex. In some embodiments, the first site-directedpolypeptide and the second site-directed polypeptide are the same. Insome embodiments, the first site-directed polypeptide and the secondsite-directed polypeptide are different.

In one aspect the disclosure provides for a vector comprising apolynucleotide sequence encoding: two or more nucleic acid-targetingnucleic acids that differ by at least one nucleotide, and asite-directed polypeptide.

In one aspect the disclosure provides a genetically modified host cellcomprising: a vector comprising a polynucleotide sequence encoding: twoor more nucleic acid-targeting nucleic acids that differ by at least onenucleotide, and a site-directed polypeptide.

In one aspect the disclosure provides a kit comprising: a vectorcomprising a polynucleotide sequence encoding: two or more nucleicacid-targeting nucleic acids that differ by at least one nucleotide, asite-directed polypeptide, and a suitable buffer. In some embodiments,the kit further comprises: a capture agent, a solid support, sequencingadaptors, and a positive control, or any combination thereof. In someembodiments, the kit further comprises instructions for use.

In one aspect the disclosure provides for a kit comprising: asite-directed polypeptide comprising reduced enzymatic activity comparedto a wild-type site-directed polypeptide, a nucleic acid-targetingnucleic acid, and a capture agent. In some embodiments, the kit furthercomprises: instructions for use. In some embodiments, the kit furthercomprises a buffer selected from the group comprising: a wash buffer, astabilization buffer, a reconstituting buffer, or a diluting buffer.

In one aspect the disclosure provides for a method for cleaving a targetnucleic acid using two or more nickases comprising: contacting a targetnucleic acid with a first complex and a second complex, wherein thefirst complex comprises a first nickase and a first nucleicacid-targeting nucleic acid, and wherein the second complex comprises asecond nickase and a second nucleic acid-targeting nucleic acid, whereinthe target nucleic acid comprises a first protospacer adjacent motif ona first strand and a second protospacer adjacent motif on a secondstrand, wherein the first nucleic acid-targeting nucleic acids isadapted to hybridize to the first protospacer adjacent motif, andwherein the second nucleic acid-targeting nucleic acid is adapted tohybridize to the second protospacer adjacent motif, and nicking thefirst and second strands of the target nucleic acid, wherein the nickinggenerates a cleaved target nucleic acid. In some embodiments, the firstand second nickase are the same. In some embodiments, the first andsecond nickase are different. In some embodiments, the first and secondnucleic acid-targeting nucleic acid are different. In some embodiments,there are less than 125 nucleotides between the first protospaceradjacent motif and the second protospacer adjacent motif. In someembodiments, the first and second protospacer adjacent motifs compriseof the sequence NGG, where N is any nucleotide. In some embodiments, thefirst or second nickase comprises at least one substantially inactivenuclease domain. In some embodiments, the first or second nickasecomprises a mutation of a conserved aspartic acid. In some embodiments,the mutation is a D10A mutation. In some embodiments, the first orsecond nickase comprises a mutation of a conserved histidine. In someembodiments, the mutation is a H840A mutation. In some embodiments,there are less than 15 nucleotides between the first and secondprotospacer adjacent motifs. In some embodiments, there are less than 10nucleotides between the first and second protospacer adjacent motifs. Insome embodiments, there are less than 5 nucleotides between the firstand second protospacer adjacent motifs. In some embodiments, the firstand second protospacer adjacent motifs are adjacent to one another. Insome embodiments, the nicking comprises the first nickase nicking thefirst strand and the second nickase nicking the second strand. In someembodiments, the nicking generates a sticky end cut. In someembodiments, the nicking generates a blunt end cut. In some embodiments,the method further comprises inserting a donor polynucleotide into thecleaved target nucleic acid.

In one aspect the disclosure provides for a composition comprising: aplurality of nucleic acid molecules, wherein each nucleic acid moleculecomprises a nucleic acid-binding protein binding site, wherein at leastone of the plurality of nucleic acid molecules encodes for a nucleicacid-targeting nucleic acid and one of the plurality of nucleic acidmolecules encodes for a site-directed polypeptide, and a fusionpolypeptide, wherein the fusion polypeptide comprises a plurality of thenucleic acid-binding proteins, wherein the plurality of nucleicacid-binding proteins are adapted to bind to their cognate nucleicacid-binding protein binding site. In some embodiments, one or more ofthe plurality of nucleic acid-binding proteins comprise a non-nativesequence. In some embodiments, the non-native sequence is located at aposition selected from the group consisting of: the N-terminus, theC-terminus, a surface accessible region, or any combination thereof. Insome embodiments, the non-native sequence encodes for a nuclearlocalization signal. In some embodiments, the plurality of nucleicacid-binding proteins are separated by a linker. In some embodiments,some of the plurality of nucleic acid-binding proteins are the samenucleic acid-binding protein. In some embodiments, all of the pluralityof nucleic acid-binding proteins are the same nucleic acid-bindingprotein. In some embodiments, the plurality of nucleic acid-bindingproteins are different nucleic acid-binding proteins. In someembodiments, the plurality of nucleic acid-binding proteins compriseRNA-binding proteins. In some embodiments, the RNA-binding proteins areselected from the group consisting of: a Type I Clustered RegularlyInterspaced Short Palindromic Repeat system endoribonuclease, a Type IIClustered Regularly Interspaced Short Palindromic Repeat systemendoribonuclease, or a Type III Clustered Regularly Interspaced ShortPalindromic Repeat system endoribonuclease, or any combination thereof.In some embodiments, the RNA-binding proteins are selected from thegroup consisting of: Cas5, Cas6, and Csy4, or any combination thereof.In some embodiments, the plurality of nucleic acid-binding proteinscomprise DNA-binding proteins. In some embodiments, the nucleicacid-binding protein binding site is configured to bind a nucleicacid-binding protein selected from the group consisting of: Type I, TypeII, and Type III Clustered Regularly Interspaced Short PalindromicRepeat system nucleic acid-binding protein, or any combination thereof.In some embodiments, the nucleic acid-binding protein binding site isconfigured to bind a nucleic acid-binding protein selected from thegroup consisting of: Cas6, Cas5, and Csy4, or any combination thereof.In some embodiments, some of the plurality of nucleic acid moleculescomprise the same nucleic acid-binding protein binding site. In someembodiments, the plurality of nucleic acid molecules comprise the samenucleic acid-binding protein binding site. In some embodiments, the noneof the plurality of nucleic acid molecules comprise the same nucleicacid-binding protein binding site. In some embodiments, thesite-directed polypeptide comprises at least 20% sequence identity to anuclease domain of Cas9. In some embodiments, the site-directedpolypeptide is Cas9. In some embodiments, at least one of the nucleicacid molecules encodes for a Clustered Regularly Interspaced ShortPalindromic Repeat endoribonuclease. In some embodiments, the ClusteredRegularly Interspaced Short Palindromic Repeat endoribonucleasecomprises at least 20% sequence similarity to Csy4. In some embodiments,the Clustered Regularly Interspaced Short Palindromic Repeatendoribonuclease comprises at least 60% sequence similarity to Csy4. Insome embodiments, the Clustered Regularly Interspaced Short PalindromicRepeat endoribonuclease is Csy4. In some embodiments, the plurality ofnucleic acid-binding proteins comprise reduced enzymatic activity. Insome embodiments, the plurality of nucleic acid-binding proteins areadapted to bind to the nucleic acid-binding protein binding site butcannot cleave the nucleic acid-binding protein binding site. In someembodiments, the nucleic acid-targeting nucleic acid comprises two RNAmolecules. In some embodiments, a portion of each of the two RNAmolecules hybridize together. In some embodiments, a first molecule ofthe two RNA molecules comprises a sequence comprising at least 60%identity to a Clustered Regularly Interspaced Short Palindromic RepeatRNA sequence over 8 contiguous nucleotides, and wherein a secondmolecule of the two RNA molecules comprises a sequence comprising atleast 60% identity to a trans-activating-Clustered Regularly InterspacedShort Palindromic Repeat RNA sequence over 6 contiguous nucleotides. Insome embodiments, the nucleic acid-targeting nucleic acid comprises onecontinuous RNA molecule wherein the continuous RNA molecule furthercomprises two domains and a linker. In some embodiments, a portion ofthe two domains of the continuous RNA molecule hybridize together. Insome embodiments, a first portion of the continuous RNA moleculecomprises a sequence comprising at least 60% identity to a ClusteredRegularly Interspaced Short Palindromic Repeat RNA sequence over 8contiguous nucleotides, and wherein a second portion of the continuousRNA molecule comprises a sequence comprising at least 60% identity to atrans-activating-Clustered Regularly Interspaced Short PalindromicRepeat RNA sequence over 6 contiguous nucleotides. In some embodiments,the nucleic acid targeting nucleic acid is adapted to hybridize with atarget nucleic acid over 6-20 nucleotides. In some embodiments, thecomposition is configured to be delivered to a cell. In someembodiments, the composition is configured to deliver equal amounts ofthe plurality of nucleic acid molecules to a cell. In some embodiments,the composition further comprises a donor polynucleotide molecule,wherein the donor polynucleotide molecule comprises a nucleicacid-binding protein binding site, wherein the binding site is bound bya nucleic acid-binding protein of the fusion polypeptide.

In one aspect the disclosure provides for a method for delivery ofnucleic acids to a subcellular location in a cell comprising:introducing into a cell a composition comprising: a plurality of nucleicacid molecules, wherein each nucleic acid molecule comprises a nucleicacid-binding protein binding site, wherein at least one of the pluralityof nucleic acid molecules encodes for a nucleic acid-targeting nucleicacid and one of the plurality of nucleic acid molecules encodes for asite-directed polypeptide, and a fusion polypeptide, wherein the fusionpolypeptide comprises a plurality of the nucleic acid-binding proteins,wherein the plurality of nucleic acid-binding proteins are adapted tobind to their cognate nucleic acid-binding protein bindingsitestoichiometrically delivering the composition to the subcellularlocation, forming a unit comprising a site-directed polypeptidetranslated from the nucleic acid molecule encoding for a site-directedpolypeptide and the nucleic acid-targeting nucleic acid, and cleaving atarget nucleic acid, wherein the site-directed polypeptide of the unitcleaves the target nucleic acid. In some embodiments, the plurality ofnucleic acid-binding proteins bind to their cognate nucleic acid-bindingprotein binding site. In some embodiments, an endoribonuclease cleavesone of the one or more nucleic acid-binding protein binding sites. Insome embodiments, an endoribonuclease cleaves the nucleic acid-bindingprotein binding sites of the nucleic acid encoding the nucleicacid-targeting nucleic acid, thereby liberating the nucleicacid-targeting nucleic acid. In some embodiments, the subcellularlocation is selected from the group consisting of: the nuclease, the ER,the golgi, the mitochondria, the cell wall, the lysosome, and thenucleus. In some embodiments, the subcellular location is the nucleus.

In one aspect the disclosure provides for a vector comprising: apolynucleotide sequence encoding a composition comprising: a pluralityof nucleic acid molecules, wherein each nucleic acid molecule comprisesa nucleic acid-binding protein binding site, wherein at least one of theplurality of nucleic acid molecules encodes for a nucleic acid-targetingnucleic acid and one of the plurality of nucleic acid molecules encodesfor a site-directed polypeptide; and a fusion polypeptide, wherein thefusion polypeptide comprises a plurality of the nucleic acid-bindingproteins, wherein the plurality of nucleic acid-binding proteins areadapted to bind to their cognate nucleic acid-binding protein bindingsite stoichiometrically delivering the composition to the subcellularlocation. In some embodiments, the vector further comprises apolynucleotide encoding a promoter. In some embodiments, the promoter isoperably linked to the polynucleotide. In some embodiments, the promoteris an inducible promoter.

In one aspect the disclosure provides for a genetically modifiedorganism comprising a vector comprising: a polynucleotide sequenceencoding for a plurality of nucleic acid molecules, wherein each nucleicacid molecule comprises a nucleic acid-binding protein binding site,wherein at least one of the plurality of nucleic acid molecules encodesfor a nucleic acid-targeting nucleic acid and one of the plurality ofnucleic acid molecules encodes for a site-directed polypeptide, and afusion polypeptide, wherein the fusion polypeptide comprises a pluralityof the nucleic acid-binding proteins, wherein the plurality of nucleicacid-binding proteins are adapted to bind to their cognate nucleicacid-binding protein binding site stoichiometrically delivering thecomposition to the subcellular location.

In one aspect the disclosure provides for a genetically modifiedorganism comprising: a composition comprising: a plurality of nucleicacid molecules, wherein each nucleic acid molecule comprises a nucleicacid-binding protein binding site, wherein at least one of the pluralityof nucleic acid molecules encodes for a nucleic acid-targeting nucleicacid and one of the plurality of nucleic acid molecules encodes for asite-directed polypeptide; and a fusion polypeptide, wherein the fusionpolypeptide comprises a plurality of the nucleic acid-binding proteins,wherein the plurality of nucleic acid-binding proteins are adapted tobind to their cognate nucleic acid-binding protein binding site.

In one aspect the disclosure provides for a kit comprising: acomposition comprising: a plurality of nucleic acid molecules, whereineach nucleic acid molecule comprises a nucleic acid-binding proteinbinding site, wherein at least one of the plurality of nucleic acidmolecules encodes for a nucleic acid-targeting nucleic acid and one ofthe plurality of nucleic acid molecules encodes for a site-directedpolypeptide, and a fusion polypeptide, wherein the fusion polypeptidecomprises a plurality of the nucleic acid-binding proteins, wherein theplurality of nucleic acid-binding proteins are adapted to bind to theircognate nucleic acid-binding protein binding site, and a buffer.

In one aspect the disclosure provides for a kit comprising: a vectorcomprising: a polynucleotide sequence encoding for a plurality ofnucleic acid molecules, wherein each nucleic acid molecule comprises anucleic acid-binding protein binding site, wherein at least one of theplurality of nucleic acid molecules encodes for a nucleic acid-targetingnucleic acid and one of the plurality of nucleic acid molecules encodesfor a site-directed polypeptide; and a fusion polypeptide, wherein thefusion polypeptide comprises a plurality of the nucleic acid-bindingproteins, wherein the plurality of nucleic acid-binding proteins areadapted to bind to their cognate nucleic acid-binding protein bindingsite stoichiometrically delivering the composition to the subcellularlocation, and a buffer. In some embodiments, the kit further comprisesinstructions for use. In some embodiments, the buffer is selected fromthe group comprising: a dilution buffer, a reconstitution buffer, and astabilization buffer, or any combination thereof.

In one aspect the disclosure provides for a donor polynucleotidecomprising: a genetic element of interest, and a reporter element,wherein the reporter element comprises a polynucleotide sequenceencoding a site-directed polypeptide, and one or more a nucleic acids,wherein the one or more nucleic acids comprises a sequence comprising atleast 50% sequence identity to a crRNA over 6 contiguous nucleotides anda sequence comprising at least 50% sequence identity to a tracrRNA over6 contiguous nucleotides. In some embodiments, the genetic element ofinterest comprises a gene. In some embodiments, the genetic element ofinterest comprises a non-coding nucleic acid selected from the groupconsisting of: a microRNA, a siRNA, and a long non-coding RNA, or anycombination thereof. In some embodiments, the genetic element ofinterest comprises a non-coding gene. In some embodiments, the geneticelement of interest comprises a non-coding nucleic acid selected fromthe group consisting of: a microRNA, a siRNA, and a long non-coding RNA,or any combination thereof. In some embodiments, the reporter elementcomprises a gene selected from the group consisting of: a gene encodinga fluorescent protein, a gene encoding a chemiluminescent protein, andan antibiotic resistance gene, or any combination thereof. In someembodiments, the reporter element comprises a gene encoding afluorescent protein. In some embodiments, the fluorescent proteincomprises green fluorescent protein. In some embodiments, the reporterelement is operably linked to a promoter. In some embodiments, thepromoter comprises an inducible promoter. In some embodiments, thepromoter comprises a tissue-specific promoter. In some embodiments, thesite-directed polypeptide comprises at least 15% amino acid sequenceidentity to a nuclease domain of Cas9. In some embodiments, thesite-directed polypeptide comprises at least 95% amino acid sequenceidentity over 10 amino acids to Cas9. In some embodiments, the nucleasedomain is selected from the group consisting of: an HNH domain, anFINE-like domain, a RuvC domain, and a RuvC-like domain, or anycombination thereof.

In one aspect the disclosure provides for an expression vectorcomprising a polynucleotide sequence encoding for a genetic element ofinterest; and a reporter element, wherein the reporter element comprisesa polynucleotide sequence encoding a site-directed polypeptide, and oneor more a nucleic acids, wherein the one or more nucleic acids comprisesa sequence comprising at least 50% sequence identity to a crRNA over 6contiguous nucleotides and a sequence comprising at least 50% sequenceidentity to a tracrRNA over 6 contiguous nucleotides.

In one aspect the disclosure provides for a genetically modified cellcomprising a donor polynucleotide comprising: a genetic element ofinterest; and a reporter element, wherein the reporter element comprisesa polynucleotide sequence encoding a site-directed polypeptide, and oneor more a nucleic acids, wherein the one or more nucleic acids comprisesa sequence comprising at least 50% sequence identity to a crRNA over 6contiguous nucleotides and a sequence comprising at least 50% sequenceidentity to a tracrRNA over 6 contiguous nucleotides.

In one aspect the disclosure provides for a kit comprising: a donorpolynucleotide comprising: a genetic element of interest; and a reporterelement, wherein the reporter element comprises a polynucleotidesequence encoding a site-directed polypeptide, and one or more a nucleicacids, wherein the one or more nucleic acids comprises a sequencecomprising at least 50% sequence identity to a crRNA over 6 contiguousnucleotides and a sequence comprising at least 50% sequence identity toa tracrRNA over 6 contiguous nucleotides; and a buffer. In someembodiments, the kit further comprises: a polypeptide comprising atleast 10% amino acid sequence identity to Cas9; and a nucleic acid,wherein the nucleic acid binds to the polypeptide and hybridizes to atarget nucleic acid. In some embodiments, the kit further comprisesinstructions for use. In some embodiments, the kit further comprises apolynucleotide encoding a polypeptide, wherein the polypeptide comprisesat last 15% amino acid sequence identity to Cas9. In some embodiments,the kit further comprises a polynucleotide encoding a nucleic acid,wherein the nucleic acid comprises a sequence comprising at least 50%sequence identity to a crRNA over 6 contiguous nucleotides and asequence comprising at least 50% sequence identity to a tracrRNA over 6contiguous nucleotides.

In one aspect the disclosure provides for a method for selecting a cellusing a reporter element and excising the reporter element from the cellcomprising: contacting a target nucleic acid with a complex comprising asite-directed polypeptide and a nucleic acid-targeting nucleic acid;cleaving the target nucleic acid with the site-directed polypeptide, togenerate a cleaved target nucleic acid; inserting the donorpolynucleotide comprising a genetic element of interest; and a reporterelement, wherein the reporter element comprises a polynucleotidesequence encoding a site-directed polypeptide, and one or more a nucleicacids, wherein the one or more nucleic acids comprises a sequencecomprising at least 50% sequence identity to a crRNA over 6 contiguousnucleotides and a sequence comprising at least 50% sequence identity toa tracrRNA over 6 contiguous nucleotides into the cleaved target nucleicacid; and selecting the cell based on the donor polynucleotide togenerate a selected cell. In some embodiments, selecting comprisesselecting the cell from a subject being treated for a disease. In someembodiments, selecting comprises selecting the cell from a subject beingdiagnosed for a disease. In some embodiments, after the selecting, thecell comprises the donor polynucleotide. In some embodiments, the methodfurther comprises excising all, some or none of the reporter element,thereby generating a second selected cell. In some embodiments, excisingcomprises contacting the 5′ end of the reporter element with a complexcomprising a site-directed polypeptide and a nucleic acid-targetingnucleic acid, wherein the complex cleaves the 5′ end. In someembodiments, excising comprises contacting the 3′ end of the reporterelement with a complex comprising a site-directed polypeptide and anucleic acid-targeting nucleic acid, wherein the complex cleaves the3′end. In some embodiments, excising comprises contacting the 5′ and 3′end of the reporter element with one or more complexes comprising asite-directed polypeptide and a nucleic acid-targeting nucleic acid,wherein the complex cleaves the 5′ and 3′end. In some embodiments, themethod further comprises screening the second selected cell. In someembodiments, screening comprises observing an absence of all or some ofthe reporter element.

In one aspect the disclosure provides for a composition comprising: anucleic acid comprising: a spacer, wherein the spacer is between 12-30nucleotides, inclusive, and wherein the spacer is adapted to hybridizeto a sequence that is 5′ to a PAM; a first duplex, wherein the firstduplex is 3′ to the spacer; a bulge, wherein the bulge comprises atleast 3 unpaired nucleotides on a first strand of the first duplex andat least 1 unpaired nucleotide on a second strand of the first duplex; alinker, wherein the linker links the first strand and the second strandof the duplex and is at least 3 nucleotides in length; a P-domain; and asecond duplex, wherein the second duplex is 3′ of the P-domain and isadapted to bind to a site directed polypeptide. In some embodiments, thesequence that is 5′ to a PAM is at least 18 nucleotides in length. Insome embodiments, the sequence that is 5′ to a PAM is adjacent to thePAM. In some embodiments, the PAM comprises 5′-NGG-3′. In someembodiments, the first duplex is adjacent to the spacer. In someembodiments, the P-domain starts from 1-5 nucleotides downstream of theduplex, comprises at least 4 nucleotides, and is adapted to hybridize tosequence selected from the group consisting of: a 5′-NGG-3′ protospaceradjacent motif sequence, a sequence comprising at least 50% identity toamino acids 1096-1225 of Cas9 from S. pyogenes, or any combinationthereof. In some embodiments, the site-directed polypeptide comprises atleast 15% identity to a nuclease domain of Cas9 from S. pyogenes. Insome embodiments, the nucleic acid is RNA. In some embodiments, thenucleic acid is an A-form RNA. In some embodiments, the first duplex isat least 6 nucleotides in length. In some embodiments, the 3 unpairednucleotides of the bulge comprise 5′-AAG-3′. In some embodiments,adjacent to the 3 unpaired nucleotides is a nucleotide that forms awobble pair with a nucleotide on the second strand of the first duplex.In some embodiments, the polypeptide binds to a region of the nucleicacid selected from the group consisting of: the first duplex, the secondduplex, and the P-domain, or any combination thereof.

In one aspect the disclosure provides for a method of modifying a targetnucleic acid comprising: contacting a target nucleic acid with acomposition comprising: a nucleic acid comprising: a spacer, wherein thespacer is between 12-30 nucleotides, inclusive, and wherein the spaceris adapted to hybridize to a sequence that is 5′ to a PAM; a firstduplex, wherein the first duplex is 3′ to the spacer; a bulge, whereinthe bulge comprises at least 3 unpaired nucleotides on a first strand ofthe first duplex and at least 1 unpaired nucleotide on a second strandof the first duplex; a linker, wherein the linker links the first strandand the second strand of the duplex and is at least 3 nucleotides inlength; a P-domain; and a second duplex, wherein the second duplex is 3′of the P-domain and is adapted to bind to a site directed polypeptide;and modifying the target nucleic acid. In some embodiments, the methodfurther comprises contacting with a site-directed polypeptide. In someembodiments, the contacting comprises contacting the spacer to thetarget nucleic acid. In some embodiments, the modifying comprisescleaving the target nucleic acid to produce a cleaved target nucleicacid. In some embodiments, the cleaving is performed by thesite-directed polypeptide. In some embodiments, the method furthercomprises inserting a donor polynucleotide into the cleaved targetnucleic acid. In some embodiments, the modifying comprises modifyingtranscription of the target nucleic acid.

In one aspect the disclosure provides for a vector comprising apolynucleotide sequence encoding a nucleic acid comprising: a spacer,wherein the spacer is between 12-30 nucleotides, inclusive, and whereinthe spacer is adapted to hybridize to a sequence that is 5′ to a PAM; afirst duplex, wherein the first duplex is 3′ to the spacer; a bulge,wherein the bulge comprises at least 3 unpaired nucleotides on a firststrand of the first duplex and at least 1 unpaired nucleotide on asecond strand of the first duplex; a linker, wherein the linker linksthe first strand and the second strand of the duplex and is at least 3nucleotides in length; a P-domain; and a second duplex, wherein thesecond duplex is 3′ of the P-domain and is adapted to bind to a sitedirected polypeptide.

In one aspect the disclosure provides for a kit comprising: acomposition comprising: a nucleic acid comprising: a spacer, wherein thespacer is between 12-30 nucleotides, inclusive, and wherein the spaceris adapted to hybridize to a sequence that is 5′ to a PAM; a firstduplex, wherein the first duplex is 3′ to the spacer; a bulge, whereinthe bulge comprises at least 3 unpaired nucleotides on a first strand ofthe first duplex and at least 1 unpaired nucleotide on a second strandof the first duplex; a linker, wherein the linker links the first strandand the second strand of the duplex and is at least 3 nucleotides inlength; a P-domain; and a second duplex, wherein the second duplex is 3′of the P-domain and is adapted to bind to a site directed polypeptide;and a buffer. In some embodiments, the kit further comprises asite-directed polypeptide. In some embodiments, the kit furthercomprises a donor polynucleotide. In some embodiments, the kit furthercomprises instructions for use.

In one aspect, the disclosure provides for a method of creating asynthetically designed nucleic acid-targeting nucleic acid comprising:designing a composition comprising: a nucleic acid comprising: a spacer,wherein the spacer is between 12-30 nucleotides, inclusive, and whereinthe spacer is adapted to hybridize to a sequence that is 5′ to a PAM; afirst duplex, wherein the first duplex is 3′ to the spacer; a bulge,wherein the bulge comprises at least 3 unpaired nucleotides on a firststrand of the first duplex and at least 1 unpaired nucleotide on asecond strand of the first duplex; a linker, wherein the linker linksthe first strand and the second strand of the duplex and is at least 3nucleotides in length; a P-domain; and a second duplex, wherein thesecond duplex is 3′ of the P-domain and is adapted to bind to a sitedirected polypeptide.

In one aspect, the disclosure provides for a pharmaceutical compositioncomprising an engineered nucleic acid-targeting nucleic acid selectedfrom the group consisting of: an engineered nucleic acid-targetingnucleic acid comprising: a mutation in a P-domain of said nucleicacid-targeting nucleic acid; an engineered nucleic acid-targetingnucleic acid comprising: a mutation in a bulge region of a nucleicacid-targeting nucleic acid

In one aspect the disclosure provides for a pharmaceutical compositioncomprising a composition selected from the group consisting of: Acomposition comprising: an engineered nucleic acid-targeting nucleicacid comprising a 3′ hybridizing extension, and a donor polynucleotide,wherein said donor polynucleotide is hybridized to said 3′ hybridizingextension; a composition comprising: an effector protein, and a nucleicacid, wherein said nucleic acid comprises: at least 50% sequenceidentity to a crRNA over 6 contiguous nucleotides, at least 50% sequenceidentity to a tracrRNA over 6 contiguous nucleotides, and a non-nativesequence, wherein said nucleic acid is adapted to bind to said effectorprotein; a composition comprising: a multiplexed genetic targetingagent, wherein said multiplexed genetic targeting agent comprises one ormore nucleic acid modules, wherein said nucleic acid module comprises anon-native sequence, and wherein said nucleic acid module is configuredto bind to a polypeptide comprising at least 10% amino acid sequenceidentity to a nuclease domain of Cas9 and wherein said nucleic acidmodule is configured to hybridize to a target nucleic acid; acomposition comprising: a modified site-directed polypeptide, whereinsaid polypeptide is modified such that it is adapted to target a secondprotospacer adjacent motif compared to a wild-type site-directedpolypeptide; a composition comprising: a modified site-directedpolypeptide, wherein said polypeptide is modified such that it isadapted to target a second nucleic acid-targeting nucleic acid comparedto a wild-type site-directed polypeptide; a composition comprising: amodified site-directed polypeptide comprising a modification in a bridgehelix as compared to SEQ ID: 8; a composition comprising: a modifiedsite-directed polypeptide comprising a modification in a highly basicpatch as compared to SEQ ID: 8; a composition comprising: a modifiedsite-directed polypeptide comprising a modification in a polymerase-likedomain as compared to SEQ ID: 8; a composition comprising: a modifiedsite-directed polypeptide comprising a modification in a bridge helix,highly basic patch, nuclease domain, and polymerase domain as comparedto SEQ ID: 8, or any combination thereof; a composition comprising: amodified site-directed polypeptide comprising a modified nuclease domainas compared to SEQ ID: 8; a composition comprising: a first complexcomprising: a first site-directed polypeptide and a first nucleicacid-targeting nucleic acid, a second complex comprising: a secondsite-directed polypeptide and a second nucleic acid-targeting nucleicacid, wherein, said first and second nucleic acid-targeting nucleicacids are different; a composition comprising: a plurality of nucleicacid molecules, wherein each nucleic acid molecule comprises a nucleicacid-binding protein binding site, wherein at least one of saidplurality of nucleic acid molecules encodes for a nucleic acid-targetingnucleic acid and one of said plurality of nucleic acid molecules encodesfor a site-directed polypeptide, and a fusion polypeptide, wherein saidfusion polypeptide comprises a plurality of said nucleic acid-bindingproteins, wherein said plurality of nucleic acid-binding proteins areadapted to bind to their cognate nucleic acid-binding protein bindingsite; and a composition comprising: a nucleic acid comprising: a spacer,wherein said spacer is between 12-30 nucleotides, inclusive, and whereinsaid spacer is adapted to hybridize to a sequence that is 5′ to a PAM, afirst duplex, wherein said first duplex is 3′ to said spacer, a bulge,wherein said bulge comprises at least 3 unpaired nucleotides on a firststrand of said first duplex and at least 1 unpaired nucleotide on asecond strand of said first duplex, a linker, wherein said linker linkssaid first strand and said second strand of said duplex and is at least3 nucleotides in length, a P-domain, and a second duplex, wherein saidsecond duplex is 3′ of said P-domain and is adapted to bind to a sitedirected polypeptide; or any combination thereof.

In one aspect, the disclosure provides for a pharmaceutical compositioncomprising a modified site-directed polypeptide comprising: a firstnuclease domain, a second nuclease domain, and an inserted nucleasedomain.

In one aspect the disclosure provides for a pharmaceutical compositioncomprising a donor polynucleotide comprising: a genetic element ofinterest, and a reporter element, wherein said reporter elementcomprises a polynucleotide sequence encoding a site-directedpolypeptide, and one or more a nucleic acids, wherein said one or morenucleic acids comprises a sequence comprising at least 50% sequenceidentity to a crRNA over 6 contiguous nucleotides and a sequencecomprising at least 50% sequence identity to a tracrRNA over 6contiguous nucleotides.

In one aspect the disclosure provides for a pharmaceutical composition avector selected from the group consisting of: a vector comprising apolynucleotide sequence encoding An engineered nucleic acid-targetingnucleic acid comprising: a mutation in a P-domain of said nucleicacid-targeting nucleic acid; a vector comprising a polynucleotidesequence encoding an engineered nucleic acid-targeting nucleic acidcomprising: a mutation in a bulge region of a nucleic acid-targetingnucleic acid; and modifying the target nucleic acid; a vector comprisinga polynucleotide sequence encoding a modified nucleic acid-targetingnucleic acid, wherein the modified nucleic acid-targeting nucleic acidcomprises a non-native sequence; a vector comprising: a polynucleotidesequence encoding: a modified nucleic acid-targeting nucleic acid,wherein the modified nucleic acid-targeting nucleic acid comprises asequence configured to bind to an effector protein, and a site-directedpolypeptide; a vector comprising: a polynucleotide sequence encoding: amodified nucleic acid-targeting nucleic acid, wherein the modifiednucleic acid-targeting nucleic acid comprises a non-native sequence, asite-directed polypeptide, and an effector protein; a vector comprisinga polynucleotide sequence encoding a multiplexed genetic targetingagent, wherein the multiplexed genetic targeting agent comprises one ormore nucleic acid modules, wherein the nucleic acid module comprises anon-native sequence, and wherein the nucleic acid module is configuredto bind to a polypeptide comprising at least 10% amino acid sequenceidentity to a nuclease domain of Cas9 and wherein the nucleic acidmodule is configured to hybridize to a target nucleic acid; a vectorcomprising a polynucleotide sequence encoding a modified site-directedpolypeptide comprising a modification in a bridge helix, highly basicpatch, nuclease domain, and polymerase domain as compared to SEQ ID: 8,or any combination thereof; a vector comprising a polynucleotidesequence encoding: two or more nucleic acid-targeting nucleic acids thatdiffer by at least one nucleotide; and a site-directed polypeptide; avector comprising: a polynucleotide sequence encoding a compositioncomprising: a plurality of nucleic acid molecules, wherein each nucleicacid molecule comprises a nucleic acid-binding protein binding site,wherein at least one of the plurality of nucleic acid molecules encodesfor a nucleic acid-targeting nucleic acid and one of the plurality ofnucleic acid molecules encodes for a site-directed polypeptide; and afusion polypeptide, wherein the fusion polypeptide comprises a pluralityof the nucleic acid-binding proteins, wherein the plurality of nucleicacid-binding proteins are adapted to bind to their cognate nucleicacid-binding protein binding sitestoichiometrically delivering thecomposition to the subcellular location; an expression vector comprisinga polynucleotide sequence encoding for a genetic element of interest;and a reporter element, wherein the reporter element comprises apolynucleotide sequence encoding a site-directed polypeptide, and one ormore a nucleic acids, wherein the one or more nucleic acids comprises asequence comprising at least 50% sequence identity to a crRNA over 6contiguous nucleotides and a sequence comprising at least 50% sequenceidentity to a tracrRNA over 6 contiguous nucleotides; and a vectorcomprising a polynucleotide sequence encoding a nucleic acid comprising:a spacer, wherein the spacer is between 12-30 nucleotides, inclusive,and wherein the spacer is adapted to hybridize to a sequence that is 5′to a PAM; a first duplex, wherein the first duplex is 3′ to the spacer;a bulge, wherein the bulge comprises at least 3 unpaired nucleotides ona first strand of the first duplex and at least 1 unpaired nucleotide ona second strand of the first duplex; a linker, wherein the linker linksthe first strand and the second strand of the duplex and is at least 3nucleotides in length; a P-domain; and a second duplex, wherein thesecond duplex is 3′ of the P-domain and is adapted to bind to a sitedirected polypeptide; or any combination thereof.

In one aspect the disclosure provides for a method of treating a diseasecomprising administering to a subject: an engineered nucleicacid-targeting comprising: a mutation in a P-domain of said nucleicacid-targeting nucleic acid; an engineered nucleic acid-targetingnucleic acid comprising: a mutation in a bulge region of a nucleicacid-targeting nucleic acid; a composition comprising: an engineerednucleic acid-targeting nucleic acid comprising a 3′ hybridizingextension, and a donor polynucleotide, wherein said donor polynucleotideis hybridized to said 3′ hybridizing extension; a compositioncomprising: an effector protein, and a nucleic acid, wherein saidnucleic acid comprises: at least 50% sequence identity to a crRNA over 6contiguous nucleotides, at least 50% sequence identity to a tracrRNAover 6 contiguous nucleotides, and a non-native sequence, wherein saidnucleic acid is adapted to bind to said effector protein; a compositioncomprising: a multiplexed genetic targeting agent, wherein saidmultiplexed genetic targeting agent comprises one or more nucleic acidmodules, wherein said nucleic acid module comprises a non-nativesequence, and wherein said nucleic acid module is configured to bind toa polypeptide comprising at least 10% amino acid sequence identity to anuclease domain of Cas9 and wherein said nucleic acid module isconfigured to hybridize to a target nucleic acid; a compositioncomprising: a modified site-directed polypeptide, wherein saidpolypeptide is modified such that it is adapted to target a secondprotospacer adjacent motif compared to a wild-type site-directedpolypeptide; a composition comprising: a modified site-directedpolypeptide, wherein said polypeptide is modified such that it isadapted to target a second nucleic acid-targeting nucleic acid comparedto a wild-type site-directed polypeptide; a composition comprising: amodified site-directed polypeptide comprising a modification in a bridgehelix as compared to SEQ ID: 8; a composition comprising: a modifiedsite-directed polypeptide comprising a modification in a highly basicpatch as compared to SEQ ID: 8; a composition comprising: a modifiedsite-directed polypeptide comprising a modification in a polymerase-likedomain as compared to SEQ ID: 8; a composition comprising: a modifiedsite-directed polypeptide comprising a modification in a bridge helix,highly basic patch, nuclease domain, and polymerase domain as comparedto SEQ ID: 8, or any combination thereof; a composition comprising: amodified site-directed polypeptide comprising a modified nuclease domainas compared to SEQ ID: 8; a composition comprising: a first complexcomprising: a first site-directed polypeptide and a first nucleicacid-targeting nucleic acid, a second complex comprising: a secondsite-directed polypeptide and a second nucleic acid-targeting nucleicacid, wherein, said first and second nucleic acid-targeting nucleicacids are different; a composition comprising: a plurality of nucleicacid molecules, wherein each nucleic acid molecule comprises a nucleicacid-binding protein binding site, wherein at least one of saidplurality of nucleic acid molecules encodes for a nucleic acid-targetingnucleic acid and one of said plurality of nucleic acid molecules encodesfor a site-directed polypeptide, and a fusion polypeptide, wherein saidfusion polypeptide comprises a plurality of said nucleic acid-bindingproteins, wherein said plurality of nucleic acid-binding proteins areadapted to bind to their cognate nucleic acid-binding protein bindingsite; a composition comprising: a nucleic acid comprising: a spacer,wherein said spacer is between 12-30 nucleotides, inclusive, and whereinsaid spacer is adapted to hybridize to a sequence that is 5′ to a PAM, afirst duplex, wherein said first duplex is 3′ to said spacer, a bulge,wherein said bulge comprises at least 3 unpaired nucleotides on a firststrand of said first duplex and at least 1 unpaired nucleotide on asecond strand of said first duplex, a linker, wherein said linker linkssaid first strand and said second strand of said duplex and is at least3 nucleotides in length, a P-domain, and a second duplex, wherein saidsecond duplex is 3′ of said P-domain and is adapted to bind to a sitedirected polypeptide; a modified site-directed polypeptide comprising: afirst nuclease domain, a second nuclease domain, and an insertednuclease domain; a donor polynucleotide comprising: a genetic element ofinterest, and a reporter element, wherein said reporter elementcomprises a polynucleotide sequence encoding a site-directedpolypeptide, and one or more a nucleic acids, wherein said one or morenucleic acids comprises a sequence comprising at least 50% sequenceidentity to a crRNA over 6 contiguous nucleotides and a sequencecomprising at least 50% sequence identity to a tracrRNA over 6contiguous nucleotides; a vector comprising a polynucleotide sequenceencoding An engineered nucleic acid-targeting nucleic acid comprising: amutation in a P-domain of said nucleic acid-targeting nucleic acid; avector comprising a polynucleotide sequence encoding an engineerednucleic acid-targeting nucleic acid comprising: a mutation in a bulgeregion of a nucleic acid-targeting nucleic acid; and modifying thetarget nucleic acid; a vector comprising a polynucleotide sequenceencoding a modified nucleic acid-targeting nucleic acid, wherein themodified nucleic acid-targeting nucleic acid comprises a non-nativesequence; a vector comprising: a polynucleotide sequence encoding: amodified nucleic acid-targeting nucleic acid, wherein the modifiednucleic acid-targeting nucleic acid comprises a sequence configured tobind to an effector protein, and a site-directed polypeptide; a vectorcomprising: a polynucleotide sequence encoding: a modified nucleicacid-targeting nucleic acid, wherein the modified nucleic acid-targetingnucleic acid comprises a non-native sequence, a site-directedpolypeptide, and an effector protein; a vector comprising apolynucleotide sequence encoding a multiplexed genetic targeting agent,wherein the multiplexed genetic targeting agent comprises one or morenucleic acid modules, wherein the nucleic acid module comprises anon-native sequence, and wherein the nucleic acid module is configuredto bind to a polypeptide comprising at least 10% amino acid sequenceidentity to a nuclease domain of Cas9 and wherein the nucleic acidmodule is configured to hybridize to a target nucleic acid; a vectorcomprising a polynucleotide sequence encoding a modified site-directedpolypeptide comprising a modification in a bridge helix, highly basicpatch, nuclease domain, and polymerase domain as compared to SEQ ID: 8,or any combination thereof; a vector comprising a polynucleotidesequence encoding: two or more nucleic acid-targeting nucleic acids thatdiffer by at least one nucleotide; and a site-directed polypeptide; avector comprising: a polynucleotide sequence encoding a compositioncomprising: a plurality of nucleic acid molecules, wherein each nucleicacid molecule comprises a nucleic acid-binding protein binding site,wherein at least one of the plurality of nucleic acid molecules encodesfor a nucleic acid-targeting nucleic acid and one of the plurality ofnucleic acid molecules encodes for a site-directed polypeptide; and afusion polypeptide, wherein the fusion polypeptide comprises a pluralityof the nucleic acid-binding proteins, wherein the plurality of nucleicacid-binding proteins are adapted to bind to their cognate nucleicacid-binding protein binding sitestoichiometrically delivering thecomposition to the subcellular location; an expression vector comprisinga polynucleotide sequence encoding for a genetic element of interest;and a reporter element, wherein the reporter element comprises apolynucleotide sequence encoding a site-directed polypeptide, and one ormore a nucleic acids, wherein the one or more nucleic acids comprises asequence comprising at least 50% sequence identity to a crRNA over 6contiguous nucleotides and a sequence comprising at least 50% sequenceidentity to a tracrRNA over 6 contiguous nucleotides; and a vectorcomprising a polynucleotide sequence encoding a nucleic acid comprising:a spacer, wherein the spacer is between 12-30 nucleotides, inclusive,and wherein the spacer is adapted to hybridize to a sequence that is 5′to a PAM; a first duplex, wherein the first duplex is 3′ to the spacer;a bulge, wherein the bulge comprises at least 3 unpaired nucleotides ona first strand of the first duplex and at least 1 unpaired nucleotide ona second strand of the first duplex; a linker, wherein the linker linksthe first strand and the second strand of the duplex and is at least 3nucleotides in length; a P-domain; and a second duplex, wherein thesecond duplex is 3′ of the P-domain and is adapted to bind to a sitedirected polypeptide; or any combination thereof. In some embodiments,the administering comprises administering comprises administering byviral delivery. In some embodiments, the administering comprisesadministering comprises administering by electroporation. In someembodiments, the administering comprises administering comprisesadministering by nanoparticle delivery. In some embodiments, theadministering comprises administering comprises administering byliposome delivery. In some embodiments, the administering comprisesadministering by a method selected from the group consisting of:intravenously, subcutaneously, intramuscularly, orally, rectally, byaerosol, parenterally, ophthalmicly, pulmonarily, transdermally,vaginally, optically, nasally, and by topical administration, or anycombination thereof. In some embodiments, the methods of the disclosureare performed in a cell selected from the group consisting of: plantcell, microbe cell, and fungi cell, or any combination thereof.

Incorporation by Reference

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A depicts an exemplary embodiment of a single guide nucleicacid-targeting nucleic acid of the disclosure.

FIG. 1B depicts an exemplary embodiment of a single guide nucleicacid-targeting nucleic acid of the disclosure.

FIG. 2 depicts an exemplary embodiment of a double guide nucleicacid-targeting nucleic acid of the disclosure.

FIG. 3 depicts an exemplary embodiment of a sequence enrichment methodof the disclosure utilizing target nucleic acid cleavage.

FIG. 4 depicts an exemplary embodiment of a sequence enrichment methodof the disclosure utilizing target nucleic acid enrichment.

FIG. 5 depicts an exemplary embodiment of a method of the disclosure fordetermining off-target binding sites of a site-directed polypeptideutilizing purification of the site-directed polypeptide.

FIG. 6 depicts an exemplary embodiment of a method of the disclosure fordetermining off-target binding sites of a site-directed polypeptideutilizing purification of the nucleic acid-targeting nucleic acid.

FIG. 7 illustrates an exemplary embodiment for an array-based sequencingmethod using a site-directed polypeptide of the disclosure.

FIG. 8 illustrates an exemplary embodiment for an array-based sequencingmethod using a site-directed polypeptide of the disclosure, whereincleaved products are sequenced.

FIG. 9 illustrates an exemplary embodiment for a next-generationsequencing-based method using a site-directed polypeptide of thedisclosure.

FIG. 10 depicts an exemplary tagged single guide nucleic acid-targetingnucleic acid.

FIG. 11 depicts an exemplary tagged double guide nucleic acid-targetingnucleic acid.

FIG. 12 illustrates an exemplary embodiment of a method of using taggednucleic acid-targeting nucleic acid with a split system (e.g., splitfluorescent system).

FIG. 13 depicts some exemplary data on the effect of a 5′ tagged nucleicacid-targeting nucleic acid on target nucleic acid cleavage.

FIG. 14 illustrates an exemplary 5′ tagged nucleic acid-targetingnucleic acid comprising a tag linker sequence between the nucleicacid-targeting nucleic acid and the tag.

FIG. 15 depicts an exemplary embodiment of a method of multiplexedtarget nucleic acid cleavage.

FIG. 16 depicts an exemplary embodiment of a method of stoichiometricdelivery of RNA nucleic acids.

FIG. 17 depicts an exemplary embodiment of a method of stoichiometricdelivery of nucleic acids.

FIG. 18 depicts an exemplary embodiment of seamless insertion of areporter element into a target nucleic acid using a site-directedpolypeptide of the disclosure.

FIG. 19 depicts an exemplary embodiment for removing a reporter elementfrom a target nucleic acid.

FIG. 20 depicts complementary portions of the nucleic acid sequences ofa pre-CRISPR nucleic acid and tracr nucleic acid sequences fromStreptococcus pyogenes SF370.

FIG. 21 depicts an exemplary secondary structure of a synthetic singleguide nucleic acid-targeting nucleic acid.

FIGS. 22A and B shows exemplary single-guide nucleic acid-targetingnucleic acid backbone variants. Nucleotides in the boxes correspond tonucleotides that have been altered relative to CRISPR sequences labeledas FL-tracr-crRNA sequence.

FIG. 23A-C shows exemplary data from an in vitro cleavage assay. Theresults demonstrate that more than one synthetic nucleic acid-targetingnucleic acid backbone sequences can support cleavage by a site-directedpolypeptide (e.g., Cas9).

FIG. 24 shows exemplary synthetic single-guide nucleic acid-targetingnucleic acid sequences containing variants in the complementaryregion/duplex. Nucleotides in the boxes correspond to nucleotides thathave been altered relative to the CRISPR sequences labeled asFL-tracr-crRNA sequence.

FIG. 25 shows exemplary variants to the single guide nucleicacid-targeting nucleic acid structure within the region 3′ to thecomplementary region/duplex. Nucleotides in the boxes correspond tonucleotides that have been altered relative to the naturally occurringS. pyogenes SF370 CRISPR nucleic acid and tracr nucleic acid sequencepairing.

FIG. 26A-B shows exemplary variants to the single guide nucleicacid-targeting nucleic acid structure within the region 3′ to thecomplementary region/duplex. Nucleotides in the boxes correspond tonucleotides that have been altered relative to the naturally occurringS. pyogenes SF370 CRISPR nucleic acid and tracr nucleic acid sequencepairing.

FIG. 27A-B shows exemplary variant nucleic acid-targeting nucleic acidstructures comprising additional hairpin sequences derived from theCRISPR repeat in Pseudomonas aeruginosa (PA14). The sequences in theboxes can bind to the ribonuclease Csy4 from PA14.

FIG. 28 shows exemplary data from an in vitro cleavage assaydemonstrating that multiple synthetic nucleic acid-targeting nucleicacid backbone sequences support Cas9 cleavage. The top and bottom gelimages represent two independent repeats of the assay.

FIG. 29 shows exemplary data from an in vitro cleavage assaydemonstrating that multiple synthetic nucleic acid-targeting nucleicacid backbone sequences support Cas9 cleavage. The top and bottom gelimages represent two independent repeats of the assay.

FIG. 30 depicts exemplary methods of the disclosure of bringing a donorpolynucleotide to a modification site in a target nucleic acid.

FIG. 31 depicts a system for storing and sharing electronic information.

FIG. 32 depicts an exemplary embodiment of two nickases generating ablunt end cut in a target nucleic acid. The site-directed modifyingpolypeptides complexed with nucleic acid-targeting nucleic acids are notshown.

FIG. 33 depicts an exemplary embodiment of staggard cutting of a targetnucleic acid using two nickases and generating sticky ends. Thesite-directed modifying polypeptides complexed with nucleicacid-targeting nucleic acids are not shown.

FIG. 34 depicts an exemplary embodiment of staggard cutting of a targetnucleic acid using two nickases and generating medium-sized sticky ends.The site-directed modifying polypeptides complexed with nucleicacid-targeting nucleic acids are not shown.

FIG. 35 illustrates a sequence alignment of Cas9 orthologues. Aminoacids with a “X” below them may be considered to be similar. Amino acidswith a “Y” below them can be considered to be highly conserved oridentical in all sequences. Amino acids residues without an “X” or a “Y”may not be conserved.

FIG. 36 shows the functionality of nucleic acid-targeting nucleic acidvariants on target nucleic acid cleavage. The variants tested in FIG. 36correspond to the variants depicted in FIG. 22, FIG. 24, and FIG. 25.

FIG. 37A-D shows in vitro cleavage assays using variant nucleicacid-targeting nucleic acids.

FIG. 38 depicts exemplary amino acid sequences of Csy4 from wild-type P.aeruginosa.

FIG. 39 depicts exemplary amino acid sequences of an enzymaticallyinactive endoribonuclease (e.g., Csy4).

FIG. 40 depicts exemplary amino acid sequences of Csy4 from P.aeruginosa.

FIG. 41A-J depicts exemplary Cas6 amino acid sequences.

FIG. 42A-C depicts exemplary Cas6 amino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, “affinity tag” can refer to either a peptide affinitytag or a nucleic acid affinity tag. Affinity tag generally refer to aprotein or nucleic acid sequence that can be bound to a molecule (e.g.,bound by a small molecule, protein, covalent bond). An affinity tag canbe a non-native sequence. A peptide affinity tag can comprise a peptide.A peptide affinity tag can be one that is able to be part of a splitsystem (e.g., two inactive peptide fragments can combine together intrans to form an active affinity tag). A nucleic acid affinity tag cancomprise a nucleic acid. A nucleic acid affinity tag can be a sequencethat can selectively bind to a known nucleic acid sequence (e.g. throughhybridization). A nucleic acid affinity tag can be a sequence that canselectively bind to a protein. An affinity tag can be fused to a nativeprotein. An affinity tag can be fused to a nucleotide sequence.Sometimes, one, two, or a plurality of affinity tags can be fused to anative protein or nucleotide sequence. An affinity tag can be introducedinto a nucleic acid-targeting nucleic acid using methods of in vitro orin vivo transcription. Nucleic acid affinity tags can include, forexample, a chemical tag, an RNA-binding protein binding sequence, aDNA-binding protein binding sequence, a sequence hybridizable to anaffinity-tagged polynucleotide, a synthetic RNA aptamer, or a syntheticDNA aptamer. Examples of chemical nucleic acid affinity tags caninclude, but are not limited to, ribo-nucleotriphosphates containingbiotin, fluorescent dyes, and digoxeginin. Examples of protein-bindingnucleic acid affinity tags can include, but are not limited to, the MS2binding sequence, the U1A binding sequence, stem-loop binding proteinsequences, the boxB sequence, the eIF4A sequence, or any sequencerecognized by an RNA binding protein. Examples of nucleic acidaffinity-tagged oligonucleotides can include, but are not limited to,biotinylated oligonucleotides, 2,4-dinitrophenyl oligonucleotides,fluorescein oligonucleotides, and primary amine-conjugatedoligonucleotides.

A nucleic acid affinity tag can be an RNA aptamer. Aptamers can include,aptamers that bind to theophylline, streptavidin, dextran B512,adenosine, guanosine, guanine/xanthine, 7-methyl-GTP, amino acidaptamers such as aptamers that bind to arginine, citrulline, valine,tryptophan, cyanocobalamine, N-methylmesoporphyrin IX, flavin, NAD, andantibiotic aptamers such as aptamers that bind to tobramycin, neomycin,lividomycin, kanamycin, streptomycin, viomycin, and chloramphenicol.

A nucleic acid affinity tag can comprise an RNA sequence that can bebound by a site-directed polypeptide. The site-directed polypeptide canbe conditionally enzymatically inactive. The RNA sequence can comprise asequence that can be bound by a member of Type I, Type II, and/or TypeIII CRISPR systems. The RNA sequence can be bound by a RAMP familymember protein. The RNA sequence can be bound by a Cas6 family memberprotein (e.g., Csy4, Cas6). The RNA sequence can be bound by a Cas5family member protein (e.g., Cas5). For example, Csy4 can bind to aspecific RNA hairpin sequence with high affinity (Kd ˜50 pM) and cancleave RNA at a site 3′ to the hairpin. The Cas5 or Cas6 family memberprotein can bind an RNA sequence that comprises at least about or atmost about 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%sequence identity and/or sequence similarity to the following nucleotidesequences:

5′-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3′; 5′-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3′5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′.5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′,5′-GUCGCCCCCCACGCGGGGGCGUGGAUUGAAAC-3′;5′-CCAGCCGCCUUCGGGCGGCUGUGUGUUGAAAC-3′;5′-GUCGCACUCUACAUGAGUGCGUGGAUUGAAAU-3′;5′-UGUCGCACCUUAUAUAGGUGCGUGGAUUGAAAU-3′; and5′-GUCGCGCCCCGCAUGGGGCGCGUGGAUUGAAA-3′,

A nucleic acid affinity tag can comprise a DNA sequence that can bebound by a site-directed polypeptide. The site-directed polypeptide canbe conditionally enzymatically inactive. The DNA sequence can comprise asequence that can be bound by a member of the Type I, Type II and/orType III CRISPR system. The DNA sequence can be bound by an Argonautprotein. The DNA sequence can be bound by a protein containing a zincfinger domain, a TALE domain, or any other DNA-binding domain.

A nucleic acid affinity tag can comprise a ribozyme sequence. Suitableribozymes can include peptidyl transferase 23S rRNA, RnaseP, Group Iintrons, Group II introns, GIR1 branching ribozyme, Leadzyme, hairpinribozymes, hammerhead ribozymes, HDV ribozymes, CPEB3 ribozymes, VSribozymes, glmS ribozyme, CoTC ribozyme, and synthetic ribozymes.

Peptide affinity tags can comprise tags that can be used for tracking orpurification (e.g., a fluorescent protein, green fluorescent protein(GFP), YFP, RFP, CFP, mCherry, tdTomato, a his tag, (e.g., a 6×His tag),a hemagglutinin (HA) tag, a FLAG tag, a Myc tag, a GST tag, a MBP tag,and chitin binding protein tag, a calmodulin tag, a V5 tag, astreptavidin binding tag, and the like).

Both nucleic acid and peptide affinity tags can comprise small moleculetags such as biotin, or digitoxin, fluorescent label tags, such as forexample, fluoroscein, rhodamin, Alexa fluor dyes, Cyanine3 dye, Cyanine5dye.

Nucleic acid affinity tags can be located 5′ to a nucleic acid (e.g., anucleic acid-targeting nucleic acid). Nucleic acid affinity tags can belocated 3′ to a nucleic acid. Nucleic acid affinity tags can be located5′ and 3′ to a nucleic acid. Nucleic acid affinity tags can be locatedwithin a nucleic acid. Peptide affinity tags can be located N-terminalto a polypeptide sequence. Peptide affinity tags can be locatedC-terminal to a polypeptide sequence. Peptide affinity tags can belocated N-terminal and C-terminal to a polypeptide sequence. A pluralityof affinity tags can be fused to a nucleic acid and/or a polypeptidesequence.

As used herein, “capture agent” can generally refer to an agent that canpurify a polypeptide and/or a nucleic acid. A capture agent can be abiologically active molecule or material (e.g. any biological substancefound in nature or synthetic, and includes but is not limited to cells,viruses, subcellular particles, proteins, including more specificallyantibodies, immunoglobulins, antigens, lipoproteins, glycoproteins,peptides, polypeptides, protein complexes, (strept)avidin-biotincomplexes, ligands, receptors, or small molecules, aptamers, nucleicacids, DNA, RNA, peptidic nucleic acids, oligosaccharides,polysaccharides, lipopolysccharides, cellular metabllites, haptens,pharmacologically active substances, alkaloids, steroids, vitamins,amino acids, and sugures). In some embodiments, the capture agent cancomprise an affinity tag. In some embodiments, a capture agent canpreferentially bind to a target polypeptide or nucleic acid of interest.Capture agents can be free floating in a mixture. Capture agents can bebound to a particle (e.g. a bead, a microbead, a nanoparticle). Captureagents can be bound to a solid or semisolid surface. In some instances,capture agents are irreversibly bound to a target. In other instances,capture agents are reversibly bound to a target (e.g. if a target can beeluted, or by use of a chemical such as imidizole).

As used herein, “Cas5” can generally refer to can refer to a polypeptidewith at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100% sequence identity and/or sequence similarity to a wild typeexemplary Cas5 polypeptide (e.g., Cas5 from D. vulgaris, and/or anysequences depicted in FIG. 42). Cas5 can generally refer to can refer toa polypeptide with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100% sequence identity and/or sequence similarity to a wildtype exemplary Cas5 polypeptide (e.g., a Cas5 from D. vulgaris). Cas5can refer to the wildtype or a modified form of the Cas5 protein thatcan comprise an amino acid change such as a deletion, insertion,substitution, variant, mutation, fusion, chimera, or any combinationthereof.

As used herein, “Cas6” can generally refer to can refer to a polypeptidewith at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100% sequence identity and/or sequence similarity to a wild typeexemplary Cas6 polypeptide (e.g., a Cas6 from T. thermophilus, and/orsequences depicted in FIG. 41). Cas6 can generally refer to can refer toa polypeptide with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100% sequence identity and/or sequence similarity to a wildtype exemplary Cas6 polypeptide (e.g., from T. thermophilus). Cas6 canrefer to the wildtype or a modified form of the Cas6 protein that cancomprise an amino acid change such as a deletion, insertion,substitution, variant, mutation, fusion, chimera, or any combinationthereof.

As used herein, “Cas9” can generally refer to a polypeptide with atleast about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%sequence identity and/or sequence similarity to a wild type exemplaryCas9 polypeptide (e.g., Cas9 from S. pyogenes (SEQ ID NO: 8, SEQ ID NO:1-256, SEQ ID NO: 795-1346). Cas9 can refer to can refer to apolypeptide with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100% sequence identity and/or sequence similarity to a wildtype exemplary Cas9 polypeptide (e.g., from S. pyogenes). Cas9 can referto the wildtype or a modified form of the Cas9 protein that can comprisean amino acid change such as a deletion, insertion, substitution,variant, mutation, fusion, chimera, or any combination thereof.

As used herein, a “cell” can generally refer to a biological cell. Acell can be the basic structural, functional and/or biological unit of aliving organism. A cell can originate from any organism having one ormore cells. Some non-limiting examples include: a prokaryotic cell,eukaryotic cell, a bacterial cell, an archaeal cell, a cell of asingle-cell eukaryotic organism, a protozoa cell, a cell from a plant(e.g. cells from plant crops, fruits, vegetables, grains, soy bean,corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin,hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers,gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), an algalcell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii,Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C.Agardh, and the like), seaweeds (e.g. kelp) a fungal cell (e.g., a yeastcell, a cell from a mushroom), an animal cell, a cell from aninvertebrate animal (e.g. fruit fly, cnidarian, echinoderm, nematode,etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile,bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, asheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.),and etcetera. Sometimes a cell is not orginating from a natural organism(e.g. a cell can be a synthetically made, sometimes termed an artificialcell).

A cell can be in vitro. A cell can be in vivo. A cell can be an isolatedcell. A cell can be a cell inside of an organism. A cell can be anorganism. A cell can be a cell in a cell culture. A cell can be one of acollection of cells. A cell can be a prokaryotic cell or derived from aprokaryotic cell. A cell can be a bacterial cell or can be derived froma bacterial cell. A cell can be an archaeal cell or derived from anarchaeal cell. A cell can be a eukaryotic cell or derived from aeukaryotic cell. A cell can be a plant cell or derived from a plantcell. A cell can be an animal cell or derived from an animal cell. Acell can be an invertebrate cell or derived from an invertebrate cell. Acell can be a vertebrate cell or derived from a vertebrate cell. A cellcan be a mammalian cell or derived from a mammalian cell. A cell can bea rodent cell or derived from a rodent cell. A cell can be a human cellor derived from a human cell. A cell can be a microbe cell or derivedfrom a microbe cell. A cell can be a fungi cell or derived from a fungicell.

A cell can be a stem cell or progenitor cell. Cells can include stemcells (e.g., adult stem cells, embryonic stem cells, iPS cells) andprogenitor cells (e.g., cardiac progenitor cells, neural progenitorcells, etc.). Cells can include mammalian stem cells and progenitorcells, including rodent stem cells, rodent progenitor cells, human stemcells, human progenitor cells, etc. Clonal cells can comprise theprogeny of a cell. A cell can comprise a target nucleic acid. A cell canbe in a living organism. A cell can be a genetically modified cell. Acell can be a host cell.

A cell can be a totipotent stem cell, however, in some embodiments ofthis disclosure, the term “cell” may be used but may not refer to atotipotent stem cell. A cell can be a plant cell, but in someembodiments of this disclosure, the term “cell” may be used but may notrefer to a plant cell. A cell can be a pluripotent cell. For example, acell can be a pluripotent hematopoietic cell that can differentiate intoother cells in the hematopoietic cell lineage but may not be able todifferentiate into any other non-hematopoetic cell. A cell may be ableto develop into a whole organism. A cell may or may not be able todevelop into a whole organism. A cell may be a whole organism.

A cell can be a primary cell. For example, cultures of primary cells canbe passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, 15times or more. Cells can be unicellular organisms. Cells can be grown inculture.

A cell can be a diseased cell. A diseased cell can have alteredmetabolic, gene expression, and/or morphologic features. A diseased cellcan be a cancer cell, a a diabetic cell, and a apoptotic cell. Adiseased cell can be a cell from a diseased subject. Exemplary diseasescan include blood disorders, cancers, metabolic disorders, eyedisorders, organ disorders, musculoskeletal disorders, cardiac disease,and the like.

If the cells are primary cells, they may be harvested from an individualby any method. For example, leukocytes may be harvested by apheresis,leukocytapheresis, density gradient separation, etc. Cells from tissuessuch as skin, muscle, bone marrow, spleen, liver, pancreas, lung,intestine, stomach, etc. can be harvested by biopsy. An appropriatesolution may be used for dispersion or suspension of the harvestedcells. Such solution can generally be a balanced salt solution, (e.g.normal saline, phosphate-buffered saline (PBS), Hank's balanced saltsolution, etc.), conveniently supplemented with fetal calf serum orother naturally occurring factors, in conjunction with an acceptablebuffer at low concentration. Buffers can include HEPES, phosphatebuffers, lactate buffers, etc. Cells may be used immediately, or theymay be stored (e.g., by freezing). Frozen cells can be thawed and can becapable of being reused. Cells can be frozen in a DMSO, serum, mediumbuffer (e.g., 10% DMSO, 50% serum, 40% buffered medium), and/or someother such common solution used to preserve cells at freezingtemperatures.

As used herein, “conditionally enzymatically inactive site-directedpolypeptide” can generally refer to a polypeptide that can bind to anucleic acid sequence in a polynucleotide in a sequence-specific manner,but may not cleave a target polynucleotide except under one or moreconditions that render the enzymatic domain active. A conditionallyenzymatically inactive site-directed polypeptide can comprise anenzymatically inactive domain that can be conditionally activated. Aconditionally enzymatically inactive site-directed polypeptide can beconditionally activated in the presence of imidazole. A conditionallyenzymatically inactive site-directed polypeptide can comprise a mutatedactive site that fails to bind its cognate ligand, resulting in anenzymatically inactive site-directed polypeptide. The mutated activesite can be designed to bind to ligand analogues, such that a ligandanalogue can bind to the mutated active site and reactive thesite-directed polypeptide. For example, ATP binding proteins cancomprise a mutated active site that can inhibit activity of the protein,yet are designed to specifically bind to ATP analogues. Binding of anATP analogue, but not ATP, can reactivate the protein. The conditionallyenzymatically inactive site-directed polypeptide can comprise one ormore non-native sequences (e.g., a fusion, an affinity tag).

As used herein, “crRNA” can generally refer to a nucleic acid with atleast about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%sequence identity and/or sequence similarity to a wild type exemplarycrRNA (e.g., a crRNA from S. pyogenes (e.g., SEQ ID NO: 569, SEQ ID NO:563-679). crRNA can generally refer to a nucleic acid with at most about5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identityand/or sequence similarity to a wild type exemplary crRNA (e.g., a crRNAfrom S. pyogenes). crRNA can refer to a modified form of a crRNA thatcan comprise an nucleotide change such as a deletion, insertion, orsubstitution, variant, mutation, or chimera. A crRNA can be a nucleicacid having at least about 60% identical to a wild type exemplary crRNA(e.g., a crRNA from S. pyogenes) sequence over a stretch of at least 6contiguous nucleotides. For example, a crRNA sequence can be at leastabout 60% identical, at least about 65% identical, at least about 70%identical, at least about 75% identical, at least about 80% identical,at least about 85% identical, at least about 90% identical, at leastabout 95% identical, at least about 98% identical, at least about 99%identical, or 100% identical, to a wild type exemplary crRNA sequence(e.g., a crRNA from S. pyogenes) over a stretch of at least 6 contiguousnucleotides.

As used herein, “CRISPR repeat” or “CRISPR repeat sequence” can refer toa minimum CRISPR repeat sequence.

As used herein, “Csy4” can generally refer to a polypeptide with at mostabout 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequenceidentity and/or sequence similarity to a wild type exemplary Csy4polypeptide (e.g., Csy4 from P. aeruginosa, see FIG. 40). Csy4 cangenerally refer to can refer to a polypeptide with at least about 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identityand/or sequence similarity to a wild type exemplary Csy4 polypeptide(e.g., Csy4 from P. aeruginosa). Csy4 can refer to the wildtype or amodified form of the Csy4 protein that can comprise an amino acid changesuch as a deletion, insertion, substitution, variant, mutation, fusion,chimera, or any combination thereof.

As used herein, “endoribonuclease” can generally refer to a polypeptidethat can cleave RNA. In some embodiments, an endoribonuclease can be asite-directed polypeptide. An endoribonuclease may be a member of aCRISPR system (e.g., Type I, Type II, Type III). Endoribonuclease canrefer to a Repeat Associated Mysterious Protein (RAMP) superfamily ofproteins (e.g., Cas6, Cas6, Cas5 families). Endoribonucleases can alsoinclude RNase A, RNase H, RNase I, RNase III family members (e.g.,Drosha, Dicer, RNase N), RNase L, RNase P, RNase PhyM, RNase T1, RNaseT2, RNase U2, RNase V1, RNase V. An endoribonuclease can refer to aconditionally enzymatically inactive endoribonuclease. Anendoribonuclease can refer to a catalytically inactive endoribonuclease.

As used herein, “donor polynucleotide” can refer to a nucleic acid thatcan be integrated into a site during genome engineering or targetnucleic acid engineering.

As used herein, “fixative” or “cross-linker” can generally refer to anagent that can fix or cross-link cells. Fixed or cross-linking cells canstabilize protein-nucleic acid complexes in the cell. Suitable fixativesand cross-linkers can include, formaldehyde, glutaraldehyde,ethanol-based fixatives, methanol-based fixatives, acetone, acetic acid,osmium tetraoxide, potassium dichromate, chromic acid, potassiumpermanganate, mercurials, picrates, formalin, paraformaldehyde,amine-reactive NHS-ester crosslinkers such asbis[sulfosuccinimidyl]suberate (BS3),3,3″-dithiobis[sulfosuccinimidylpropionate] (DTSSP), ethylene glycolbis[sulfosuccinimidylsuccinate (sulfo-EGS), disuccinimidyl glutarate(DSG), dithiobis[succinimidyl propionate] (DSP), disuccinimidyl suberate(DSS), ethylene glycol bis[succinimidylsuccinate] (EGS),NHS-ester/diazirine crosslinkers such as NHS-diazirine,NHS-LC-diazirine, NHS-SS-diazirine, sulfo-NHS-diazirine,sulfo-NHS-LC-diazirine, and sulfo-NHS-SS-diazirine.

As used herein, “fusion” can refer to a protein and/or nucleic acidcomprising one or more non-native sequences (e.g., moieties). A fusioncan comprise one or more of the same non-native sequences. A fusion cancomprise one or more of different non-native sequences. A fusion can bea chimera. A fusion can comprise a nucleic acid affinity tag. A fusioncan comprise a barcode. A fusion can comprise a peptide affinity tag. Afusion can provide for subcellular localization of the site-directedpolypeptide (e.g., a nuclear localization signal (NLS) for targeting tothe nucleus, a mitochondrial localization signal for targeting to themitochondria, a chloroplast localization signal for targeting to achloroplast, an endoplasmic reticulum (ER) retention signal, and thelike). A fusion can provide a non-native sequence (e.g., affinity tag)that can be used to track or purify. A fusion can be a small moleculesuch as biotin or a dye such as alexa fluor dyes, Cyanine3 dye, Cyanine5dye. The fusion can provide for increased or decreased stability.

In some embodiments, a fusion can comprise a detectable label, includinga moiety that can provide a detectable signal. Suitable detectablelabels and/or moieties that can provide a detectable signal can include,but are not limited to, an enzyme, a radioisotope, a member of aspecific binding pair; a fluorophore; a fluorescent protein; a quantumdot; and the like.

A fusion can comprise a member of a FRET pair. FRET pairs(donor/acceptor) suitable for use can include, but are not limited to,EDANS/fluorescein, IAEDANS/fluorescein,fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/DABCYL,fluorescein/QSY-7, fluorescein/LC Red 640, fluorescein/Cy 5.5 andfluorescein/LC Red 705.

A fluorophore/quantum dot donor/acceptor pair can be used as a fusion.Suitable fluorophores (“fluorescent label”) can include any moleculethat may be detected via its inherent fluorescent properties, which caninclude fluorescence detectable upon excitation. Suitable fluorescentlabels can include, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 andOregon green.

A fusion can comprise an enzyme. Suitable enzymes can include, but arenot limited to, horse radish peroxidase, luciferase, beta-galactosidase,and the like.

A fusion can comprise a fluorescent protein. Suitable fluorescentproteins can include, but are not limited to, a green fluorescentprotein (GFP), (e.g., a GFP from Aequoria victoria, fluorescent proteinsfrom Anguilla japonica, or a mutant or derivative thereof), a redfluorescent protein, a yellow fluorescent protein, any of a variety offluorescent and colored proteins.

A fusion can comprise a nanoparticle. Suitable nanoparticles can includefluorescent or luminescent nanoparticles, and magnetic nanoparticles.Any optical or magnetic property or characteristic of thenanoparticle(s) can be detected.

A fusion can comprise quantum dots (QDs). QDs can be rendered watersoluble by applying coating layers comprising a variety of differentmaterials. For example, QDs can be solubilized using amphiphilicpolymers. Exemplary polymers that have been employed can includeoctylamine-modified low molecular weight polyacrylic acid,polyethylene-glycol (PEG)-derivatized phospholipids, polyanhydrides,block copolymers, etc. QDs can be conjugated to a polypeptide via any ofa number of different functional groups or linking agents that can bedirectly or indirectly linked to a coating layer. QDs with a widevariety of absorption and emission spectra are commercially available,e.g., from Quantum Dot Corp. (Hayward Calif.; now owned by Invitrogen)or from Evident Technologies (Troy, N.Y.). For example, QDs having peakemission wavelengths of approximately 525, 535, 545, 565, 585, 605, 655,705, and 800 nm are available. Thus the QDs can have a range ofdifferent colors across the visible portion of the spectrum and in somecases even beyond.

Suitable radioisotopes can include, but are not limited to ¹⁴C, ³H, ³²P,³³P, ³⁵S, and ¹²⁵I.

As used herein, “genetically modified cell” can generally refer to acell that has been genetically modified. Some non-limiting examples ofgenetic modifications can include: insertions, deletions, inversions,translocations, gene fusions, or changing one or more nucleotides. Agenetically modified cell can comprise a target nucleic acid with anintroduced double strand break (e.g., DNA break). A genetically modifiedcell can comprise an exogenously introduced nucleic acid (e.g., avector). A genetically modified cell can comprise an exogenouslyintroduced polypeptide of the disclosure and/or nucleic acid of thedisclosure. A genetically modified cell can comprise a donorpolynucleotide. A genetically modified cell can comprise an exogenousnucleic acid integrated into the genome of the genetically modifiedcell. A gentetically modified cell can comprise a deletion of DNA. Agenetically modified cell can also refer to a cell with modifiedmitochondrial or chloroplast DNA.

As used herein, “genome engineering” can refer to a process of modifyinga target nucleic acid. Genome engineering can refer to the integrationof non-native nucleic acid into native nucleic acid. Genome engineeringcan refer to the targeting of a site-directed polypeptide and a nucleicacid-targeting nucleic acid to a target nucleic acid, without anintegration or a deletion of the target nucleic acid. Genome engineeringcan refer to the cleavage of a target nucleic acid, and the rejoining ofthe target nucleic acid without an integration of an exogenous sequencein the target nucleic acid, or a deletion in the target nucleic acid.The native nucleic acid can comprise a gene. The non-native nucleic acidcan comprise a donor polynucleotide. In the methods of the disclosure,site-directed polypeptides (e.g., Cas9) can introduce double-strandedbreaks in nucleic acid, (e.g. genomic DNA). The double-stranded breakcan stimulate a cell's endogenous DNA-repair pathways (e.g. homologousrecombination (HR) and/or non-homologous end joining (NHEJ), or A-NHEJ(alternative non-homologous end-joining)). Mutations, deletions,alterations, and integrations of foreign, exogenous, and/or alternativenucleic acid can be introduced into the site of the double-stranded DNAbreak.

As used herein, the term “isolated” can refer to a nucleic acid orpolypeptide that, by the hand of a human, exists apart from its nativeenvironment and is therefore not a product of nature. Isolated can meansubstantially pure. An isolated nucleic acid or polypeptide can exist ina purified form and/or can exist in a non-native environment such as,for example, in a transgenic cell.

As used herein, “non-native” can refer to a nucleic acid or polypeptidesequence that is not found in a native nucleic acid or protein.Non-native can refer to affinity tags. Non-native can refer to fusions.Non-native can refer to a naturally occurring nucleic acid orpolypeptide sequence that comprises mutations, insertions and/ordeletions. A non-native sequence may exhibit and/or encode for anactivity (e.g., enzymatic activity, methyltransferase activity,acetyltransferase activity, kinase activity, ubiquitinating activity,etc.) that can also be exhibited by the nucleic acid and/or polypeptidesequence to which the non-native sequence is fused. A non-native nucleicacid or polypeptide sequence may be linked to a naturally-occurringnucleic acid or polypeptide sequence (or a variant thereof) by geneticengineering to generate a chimeric nucleic acid and/or polypeptidesequence encoding a chimeric nucleic acid and/or polypeptide. Anon-native sequence can refer to a 3′ hybridizing extension sequence.

As used herein, a “nucleic acid” can generally refer to a polynucleotidesequence, or fragment thereof. A nucleic acid can comprise nucleotides.A nucleic acid can be exogenous or endogenous to a cell. A nucleic acidcan exist in a cell-free environment. A nucleic acid can be a gene orfragment thereof. A nucleic acid can be DNA. A nucleic acid can be RNA.A nucleic acid can comprise one or more analogs (e.g. altered backgone,sugar, or nucleobase). Some non-limiting examples of analogs include:5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos,locked nucleic acids, glycol nucleic acids, threose nucleic acids,dideoxynucleotides, cordycepin, 7-deaza-GTP, florophores (e.g. rhodamineor flurescein linked to the sugar), thiol containing nucleotides, biotinlinked nucleotides, fluorescent base analogs, CpG islands,methyl-7-guanosine, methylated nucleotides, inosine, thiouridine,pseudourdine, dihydrouridine, queuosine, and wyosine.

As used herein, a “nucleic acid sample” can generally refer to a samplefrom a biological entity. A nucleic acid sample can comprise nucleicacid. The nucleic acid from the nucleic acid sample can be purifiedand/or enriched. The nucleic acid sample may show the nature of thewhole. Nucleic acid samples can come from various sources. Nucleic acidsamples can come from one or more individuals. One or more nucleic acidsamples can come from the same individual. One non limiting examplewould be if one sample came from an individual's blood and a secondsample came from an individual's tumor biopsy. Examples of nucleic acidsamples can include but are not limited to, blood, serum, plasma, nasalswab or nasopharyngeal wash, saliva, urine, gastric fluid, spinal fluid,tears, stool, mucus, sweat, earwax, oil, glandular secretion, cerebralspinal fluid, tissue, semen, vaginal fluid, interstitial fluids,including interstitial fluids derived from tumor tissue, ocular fluids,spinal fluid, throat swab, cheeck swab, breath, hair, finger nails,skin, biopsy, placental fluid, amniotic fluid, cord blood, emphaticfluids, cavity fluids, sputum, pus, micropiota, meconium, breast milk,buccal samples, nasopharyngeal wash, other excretions, or anycombination thereof. Nucleic acid samples can originate from tissues.Examples of tissue samples may include but are not limited to,connective tissue, muscle tissue, nervous tissue, epithelial tissue,cartilage, cancerous or tumor sample, bone marrow, or bone. The nucleicacid sample may be provided from a human or animal. The nucleic acidsample may be provided from a mammal, vertebrate, such as murines,simians, humans, farm animals, sport animals, or pets. The nucleic acidsample may be collected from a living or dead subject. The nucleic acidsample may be collected fresh from a subject or may have undergone someform of pre-processing, storage, or transport.

A nucleic acid sample can comprise a target nucleic acid. A nucleic acidsample can originate from cell lysate. The cell lysate can originatefrom a cell.

As used herein, “nucleic acid-targeting nucleic acid” can refer to anucleic acid that can hybridize to another nucleic acid. A nucleicacid-targeting nucleic acid can be RNA. A nucleic acid-targeting nucleicacid can be DNA. The nucleic acid-targeting nucleic acid can beprogrammed to bind to a sequence of nucleic acid site-specifically. Thenucleic acid to be targeted, or the target nucleic acid, can comprisenucleotides. The nucleic acid-targeting nucleic acid can comprisenucleotides. A portion of the target nucleic acid can be complementaryto a portion of the nucleic acid-targeting nucleic acid. A nucleicacid-targeting nucleic acid can comprise a polynucleotide chain and canbe called a “single guide nucleic acid” (i.e. a “single guide nucleicacid-targeting nucleic acid”). A nucleic acid-targeting nucleic acid cancomprise two polynucleotide chains and can be called a “double guidenucleic acid” (i.e. a “double guide nucleic acid-targeting nucleicacid”). If not otherwise specified, the term “nucleic acid-targetingnucleic acid” can be inclusive, referring to both single guide nucleicacids and double guide nucleic acids.

A nucleic acid-targeting nucleic acid can comprise a segment that can bereferred to as a “nucleic acid-targeting segment” or a “nucleicacid-targeting sequence,” A nucleic acid-targeting nucleic acid cancomprise a segment that can be referred to as a “protein bindingsegment” or “protein binding sequence.”

A nucleic acid-targeting nucleic acid can comprise one or moremodifications (e.g., a base modification, a backbone modification), toprovide the nucleic acid with a new or enhanced feature (e.g., improvedstability). A nucleic acid-targeting nucleic acid can comprise a nucleicacid affinity tag. A nucleoside can be a base-sugar combination. Thebase portion of the nucleoside can be a heterocyclic base. The two mostcommon classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides can be nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to the 2′, the 3′, or the 5′ hydroxylmoiety of the sugar. In forming nucleic acid-targeting nucleic acids,the phosphate groups can covalently link adjacent nucleosides to oneanother to form a linear polymeric compound. In turn, the respectiveends of this linear polymeric compound can be further joined to form acircular compound; however, linear compounds are generally suitable. Inaddition, linear compounds may have internal nucleotide basecomplementarity and may therefore fold in a manner as to produce a fullyor partially double-stranded compound. Within nucleic acid-targetingnucleic acids, the phosphate groups can commonly be referred to asforming the internucleoside backbone of the nucleic acid-targetingnucleic acid. The linkage or backbone of the nucleic acid-targetingnucleic acid can be a 3′ to 5′ phosphodiester linkage.

A nucleic acid-targeting nucleic acid can comprise a modified backboneand/or modified internucleoside linkages. Modified backbones can includethose that retain a phosphorus atom in the backbone and those that donot have a phosphorus atom in the backbone.

Suitable modified nucleic acid-targeting nucleic acid backbonescontaining a phosphorus atom therein can include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates such as 3′-alkylene phosphonates, 5′-alkylene phosphonates,chiral phosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates, and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs, and those havinginverted polarity wherein one or more internucleotide linkages is a 3′to 3′, a 5′ to 5′ or a 2′ to 2′ linkage. Suitable nucleic acid-targetingnucleic acids having inverted polarity can comprise a single 3′ to 3′linkage at the 3′-most internucleotide linkage (i.e. a single invertednucleoside residue in which the nucleobase is missing or has a hydroxylgroup in place thereof). Various salts (e.g., potassium chloride orsodium chloride), mixed salts, and free acid forms can also be included.

A nucleic acid-targeting nucleic acid can comprise one or morephosphorothioate and/or heteroatom internucleoside linkages, inparticular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (i.e. a methylene(methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— (wherein the nativephosphodiester internucleotide linkage is represented as—O—P(═O)(OH)—O—CH₂—).

A nucleic acid-targeting nucleic acid can comprise a morpholino backbonestructure. For example, a nucleic acid can comprise a 6-memberedmorpholino ring in place of a ribose ring. In some of these embodiments,a phosphorodiamidate or other non-phosphodiester internucleoside linkagecan replace a phosphodiester linkage.

A nucleic acid-targeting nucleic acid can comprise polynucleotidebackbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These can include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

A nucleic acid-targeting nucleic acid can comprise a nucleic acidmimetic. The term “mimetic” can be intended to include polynucleotideswherein only the furanose ring or both the furanose ring and theinternucleotide linkage are replaced with non-furanose groups,replacement of only the furanose ring can also be referred as being asugar surrogate. The heterocyclic base moiety or a modified heterocyclicbase moiety can be maintained for hybridization with an appropriatetarget nucleic acid. One such nucleic acid can be a peptide nucleic acid(PNA). In a PNA, the sugar-backbone of a polynucleotide can be replacedwith an amide containing backbone, in particular an aminoethylglycinebackbone. The nucleotides can be retained and are bound directly orindirectly to aza nitrogen atoms of the amide portion of the backbone.The backbone in PNA compounds can comprise two or more linkedaminoethylglycine units which gives PNA an amide containing backbone.The heterocyclic base moieties can be bound directly or indirectly toaza nitrogen atoms of the amide portion of the backbone.

A nucleic acid-targeting nucleic acid can comprise linked morpholinounits (i.e. morpholino nucleic acid) having heterocyclic bases attachedto the morpholino ring Linking groups can link the morpholino monomericunits in a morpholino nucleic acid. Non-ionic morpholino-basedoligomeric compounds can have less undesired interactions with cellularproteins. Morpholino-based polynucleotides can be nonionic mimics ofnucleic acid-targeting nucleic acids. A variety of compounds within themorpholino class can be joined using different linking groups. A furtherclass of polynucleotide mimetic can be referred to as cyclohexenylnucleic acids (CeNA). The furanose ring normally present in a nucleicacid molecule can be replaced with a cyclohexenyl ring. CeNA DMTprotected phosphoramidite monomers can be prepared and used foroligomeric compound synthesis using phosphoramidite chemistry. Theincorporation of CeNA monomers into a nucleic acid chain can increasethe stability of a DNA/RNA hybrid. CeNA oligoadenylates can formcomplexes with nucleic acid complements with similar stability to thenative complexes. A further modification can include Locked NucleicAcids (LNAs) in which the 2′-hydroxyl group is linked to the 4′ carbonatom of the sugar ring thereby forming a 2′-C,4′-C-oxymethylene linkagethereby forming a bicyclic sugar moiety. The linkage can be a methylene(—CH2-), group bridging the 2′ oxygen atom and the 4′ carbon atomwherein n is 1 or 2. LNA and LNA analogs can display very high duplexthermal stabilities with complementary nucleic acid (Tm=+3 to +10° C.),stability towards 3′-exonucleolytic degradation and good solubilityproperties.

A nucleic acid-targeting nucleic acid can comprise one or moresubstituted sugar moieties. Suitable polynucleotides can comprise asugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-,S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein thealkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly suitable areO((CH2)nO)mCH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH2, O(CH₂)₁CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON((CH₂)_(n)CH₃)₂, where n and m are from1 to about 10. A sugar substituent group can be selected from: C₁ to C₁₀lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an nucleic acid-targeting nucleic acid, ora group for improving the pharmacodynamic properties of an nucleicacid-targeting nucleic acid, and other substituents having similarproperties. A suitable modification can include 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE i.e., analkoxyalkoxy group). A further suitable modification can include2′-dimethylaminooxyethoxy, (i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE), and 2′-dimethylaminoethoxyethoxy (also known as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH2-O—CH₂—N(CH₃)₂.

Other suitable sugar substituent groups can include methoxy (—O—CH₃),aminopropoxy CH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl CH₂—CH═CH₂)and fluoro (F). 2′-sugar substituent groups may be in the arabino (up)position or ribo (down) position. A suitable 2′-arabino modification is2′-F. Similar modifications may also be made at other positions on theoligomeric compound, particularly the 3′ position of the sugar on the 3′terminal nucleoside or in 2′-5′ linked nucleotides and the 5′ positionof 5′ terminal nucleotide. Oligomeric compounds may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar.

A nucleic acid-targeting nucleic acid may also include nucleobase (oftenreferred to simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases can include the purinebases, (e.g. adenine (A) and guanine (G)), and the pyrimidine bases,(e.g. thymine (T), cytosine (C) and uracil (U)). Modified nucleobasescan include other synthetic and natural nucleobases such as5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl (—C═C—CH3) uracil and cytosine and otheralkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Modified nucleobases can include tricyclic pyrimidinessuch as phenoxazinecytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindolecytidine (Hpyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one).

Heterocyclic base moieties can include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Nucleobases can be useful for increasing the binding affinity of apolynucleotide compound. These can include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions can increase nucleic acid duplexstability by 0.6-1.2° C. and can be suitable base substitutions (e.g.,when combined with 2′-O-methoxyethyl sugar modifications).

A modification of a nucleic acid-targeting nucleic acid can comprisechemically linking to the nucleic acid-targeting nucleic acid one ormore moieties or conjugates that can enhance the activity, cellulardistribution or cellular uptake of the nucleic acid-targeting nucleicacid. These moieties or conjugates can include conjugate groupscovalently bound to functional groups such as primary or secondaryhydroxyl groups. Conjugate groups can include, but are not limited to,intercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that can enhance the pharmacokinetic propertiesof oligomers. Conjugate groups can include, but are not limited to,cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic propertiesinclude groups that improve uptake, enhance resistance to degradation,and/or strengthen sequence-specific hybridization with the targetnucleic acid. Groups that can enhance the pharmacokinetic propertiesinclude groups that improve uptake, distribution, metabolism orexcretion of a nucleic acid. Conjugate moieties can include but are notlimited to lipid moieties such as a cholesterol moiety, cholic acid athioether, (e.g., hexyl-S-tritylthiol), a thiocholesterol, an aliphaticchain (e.g., dodecandiol or undecyl residues), a phospholipid (e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

A modification may include a “Protein Transduction Domain” or PTD (i.e.a cell penetrating peptide (CPP)). The PTD can refer to a polypeptide,polynucleotide, carbohydrate, or organic or inorganic compound thatfacilitates traversing a lipid bilayer, micelle, cell membrane,organelle membrane, or vesicle membrane. A PTD can be attached toanother molecule, which can range from a small polar molecule to a largemacromolecule and/or a nanoparticle, and can facilitate the moleculetraversing a membrane, for example going from extracellular space tointracellular space, or cytosol to within an organelle. A PTD can becovalently linked to the amino terminus of a polypeptide. A PTD can becovalently linked to the carboxyl terminus of a polypeptide. A PTD canbe covalently linked to a nucleic acid. Exemplary PTDs can include, butare not limited to, a minimal peptide protein transduction domain; apolyarginine sequence comprising a number of arginines sufficient todirect entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50arginines), a VP22 domain, a Drosophila Antennapedia proteintransduction domain, a truncated human calcitonin peptide, polylysine,and transportan, arginine homopolymer of from 3 arginine residues to 50arginine residues. The PTD can be an activatable CPP (ACPP). ACPPs cancomprise a polycationic CPP (e.g., Arg9 or “R9”) connected via acleavable linker to a matching polyanion (e.g., G1u9 or “E9”), which canreduce the net charge to nearly zero and thereby inhibits adhesion anduptake into cells. Upon cleavage of the linker, the polyanion can bereleased, locally unmasking the polyarginine and its inherentadhesiveness, thus “activating” the ACPP to traverse the membrane.

“Nucleotide” can generally refer to a base-sugar-phosphate combination.A nucleotide can comprise a synthetic nucleotide. A nucleotide cancomprise a synthetic nucleotide analog. Nucleotides can be monomericunits of a nucleic acid sequence (e.g. deoxyribonucleic acid (DNA) andribonucleic acid (RNA)). The term nucleotide can include ribonucleosidetriphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP),cytosine triphosphate (CTP), guanosine triphosphate (GTP) anddeoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP,dTTP, or derivatives thereof. Such derivatives can include, for example,[aS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives thatconfer nuclease resistance on the nucleic acid molecule containing them.The term nucleotide as used herein can refer to dideoxyribonucleosidetriphosphates (ddNTPs) and their derivatives. Illustrative examples ofdideoxyribonucleoside triphosphates can include, but are not limited to,ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide may be unlabeled ordetectably labeled by well-known techniques. Labeling can also becarried out with quantum dots. Detectable labels can include, forexample, radioactive isotopes, fluorescent labels, chemiluminescentlabels, bioluminescent labels and enzyme labels. Fluorescent labels ofnucleotides may include but are not limited fluorescein,5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanineand 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Specificexamples of fluorescently labeled nucleotides can include [R6G]dUTP,[TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP,[FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP,[dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from PerkinElmer, Foster City, Calif. FluoroLink DeoxyNucleotides, FluoroLinkCy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLinkCy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, ArlingtonHeights, Ill.; Fluorescein-15-dATP, Fluorescein-12-dUTP,Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP,Fluorescein-12-UTP, and Fluorescein-15-2′-dATP available from BoehringerMannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides,BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP,BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, CascadeBlue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP,fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP,Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP,tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, andTexas Red-12-dUTP available from Molecular Probes, Eugene, Oreg.Nucleotides can also be labeled or marked by chemical modification. Achemically-modified single nucleotide can be biotin-dNTP. Somenon-limiting examples of biotinylated dNTPs can include, biotin-dATP(e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP,biotin-14-dCTP), and biotin-dUTP (e.g. biotin-11-dUTP, biotin-16-dUTP,biotin-20-dUTP).

As used herein, “P-domain” can refer to a region in a nucleicacid-targeting nucleic acid. The P-domain can interact with aprotospacer adjacent motif (PAM), site-directed polypeptide, and/ornucleic acid-targeting nucleic acid. A P-domain can interact directly orindirectly with a protospacer adjacent motif (PAM), site-directedpolypeptide, and/or nucleic acid-targeting nucleic acid nucleic acid. Asused herein, the terms “PAM interacting region” “anti-repeat adjacentregion” and “P-domain” can be used interchangeably.

As used here, “purified” can refer to a molecule (e.g., site-directedpolypeptide, nucleic acid-targeting nucleic acid) that comprises atleast 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% of thecomposition. For example, a sample that comprises 10% of a site-directedpolypeptide, but after a purification step comprises 60% of thesite-directed polypeptide, then the sample can be said to be purified. Apurified sample can refer to an enriched sample, or a sample that hasundergone methods to remove particles other than the particle ofinterest.

As used herein, “reactivation agent” can generally refer to any agentthat can convert an enzymatically inactive polypeptide into anenzymatically active polypeptide. Imidazole can be a reactivation agent.A ligand analogue can be a reactivation agent.

As used herein, “recombinant” can refer to sequence that originates froma source foreign to the particular host (e.g., cell) or, if from thesame source, is modified from its original form. A recombinant nucleicacid in a cell can include a nucleic acid that is endogenous to theparticular cell but has been modified through, for example, the use ofsite-directed mutagenesis. The term can include non-naturally occurringmultiple copies of a naturally occurring DNA sequence. Thus, the termcan refer to a nucleic acid that is foreign or heterologous to the cell,or homologous to the cell but in a position or form within the cell inwhich the nucleic acid is not ordinarily found. Similarly, when used inthe context of a polypeptide or amino acid sequence, an exogenouspolypeptide or amino acid sequence can be a polypeptide or amino acidsequence that originates from a source foreign to the particular cellor, if from the same source, is modified from its original form.

As used herein, “site-directed polypeptides” can generally refer tonucleases, site-directed nucleases, endoribonucleases, conditionallyenzymatically inactive endoribonucleases, Argonauts, and nucleicacid-binding proteins. A site-directed polypeptide or protein caninclude nucleases such as homing endonucleases such as PI-TliII, H-DreI,I-DmoI and I-CreI, I-SceI, LAGLIDADG family nucleases, meganucleases,GIY-YIG family nucleases, His-Cys box family nucleases, Vsr-likenucleases, endoribonucleases, exoribonucleases, endonucleases, andexonucleases. A site-directed polypeptide can refer to a Cas gene memberof the Type I, Type II, Type III, and/or Type U CRISPR/Cas systems. Asite-directed polypeptide can refer to a member of the Repeat AssociatedMysterious Protein (RAMP) superfamily (e.g., Cas5, Cas6 subfamilies). Asite-directed polypeptide can refer to an Argonaute protein.

A site-directed polypeptide can be a type of protein. A site-directedpolypeptide can refer to an nuclease. A site-directed polypeptide canrefer to an endoribonuclease. A site-directed polypeptide can refer toany modified (e.g., shortened, mutated, lengthened) polypeptide sequenceor homologue of the site-directed polypeptide. A site-directedpolypeptide can be codon optimized. A site-directed polypeptide can be acodon-optimized homologue of a site-directed polypeptide. Asite-directed polypeptide can be enzymatically inactive, partiallyactive, constitutively active, fully active, inducible active and/ormore active, (e.g. more than the wild type homologue of the protein orpolypeptide.). A site-directed polypeptide can be Cas9. A site-directedpolypeptide can be Csy4. A site-directed polypeptide can be Cas5 or aCas5 family member. A site-directed polypeptide can be Cas6 or a Cas6family member.

In some instances, the site-directed polypeptide (e.g., variant,mutated, enzymatically inactive and/or conditionally enzymaticallyinactive site-directed polypeptide) can target nucleic acid. Thesite-directed polypeptide (e.g., variant, mutated, enzymaticallyinactive and/or conditionally enzymatically inactive endoribonuclease)can target RNA. Endoribonucleases that can target RNA can includemembers of other CRISPR subfamilies such as Cas6 and Cas5.

As used herein, the term “specific” can refer to interaction of twomolecules where one of the molecules through, for example chemical orphysical means, specifically binds to the second molecule. Exemplaryspecific binding interactions can refer to antigen-antibody binding,avidin-biotin binding, carbohydrates and lectins, complementary nucleicacid sequences (e.g., hybridizing), complementary peptide sequencesincluding those formed by recombinant methods, effector and receptormolecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes,and the like. “Non-specific” can refer to an interaction between twomolecules that is not specific.

As used herein, “solid support” can generally refer to any insoluble, orpartially soluble material. A solid support can refer to a test strip, amulti-well dish, and the like. The solid support can comprise a varietyof substances (e.g., glass, polystyrene, polyvinyl chloride,polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose,natural and modified celluloses, polyacrylamides, agaroses, andmagnetite) and can be provided in a variety of forms, including agarosebeads, polystyrene beads, latex beads, magnetic beads, colloid metalparticles, glass and/or silicon chips and surfaces, nitrocellulosestrips, nylon membranes, sheets, wells of reaction trays (e.g.,multi-well plates), plastic tubes, etc. A solid support can be solid,semisolid, a bead, or a surface. The support can mobile in a solution orcan be immobile. A solid support can be used to capture a polypeptide. Asolid support can comprise a capture agent.

As used herein, “target nucleic acid” can generally refer to a nucleicacid to be used in the methods of the disclosure. A target nucleic acidcan refer to a chromosomal sequence or an extrachromosomal sequence,(e.g. an episomal sequence, a minicircle sequence, a mitochondrialsequence, a chloroplast sequence, etc.). A target nucleic acid can beDNA. A target nucleic acid can be RNA. A target nucleic acid can hereinbe used interchangeably with “polynucleotide”, “nucleotide sequence”,and/or “target polynucleotide”. A target nucleic acid can be a nucleicacid sequence that may not be related to any other sequence in a nucleicacid sample by a single nucleotide substitution. A target nucleic acidcan be a nucleic acid sequence that may not be related to any othersequence in a nucleic acid sample by a 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotide substitutions. In some embodiments, the substitution cannotoccur within 5, 10, 15, 20, 25, 30, or 35 nucleotides of the 5′ end of atarget nucleic acid. In some embodiments, the substitution cannot occurwithin 5, 10, 15, 20, 25, 30, 35 nucleotides of the 3′ end of a targetnucleic acid.

As used herein, “tracrRNA” can generally refer to a nucleic acid with atleast about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%sequence identity and/or sequence similarity to a wild type exemplarytracrRNA sequence (e.g., a tracrRNA from S. pyogenes (SEQ ID 433), SEQIDs 431-562). tracrRNA can refer to a nucleic acid with at most about5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identityand/or sequence similarity to a wild type exemplary tracrRNA sequence(e.g., a tracrRNA from S. pyogenes). tracrRNA can refer to a modifiedform of a tracrRNA that can comprise an nucleotide change such as adeletion, insertion, or substitution, variant, mutation, or chimera. AtracrRNA can refer to a nucleic acid that can be at least about 60%identical to a wild type exemplary tracrRNA (e.g., a tracrRNA from S.pyogenes) sequence over a stretch of at least 6 contiguous nucleotides.For example, a tracrRNA sequence can be at least about 60% identical, atleast about 65% identical, at least about 70% identical, at least about75% identical, at least about 80% identical, at least about 85%identical, at least about 90% identical, at least about 95% identical,at least about 98% identical, at least about 99% identical, or 100%identical, to a wild type exemplary tracrRNA (e.g., a tracrRNA from S.pyogenes) sequence over a stretch of at least 6 contiguous nucleotides.A tracrRNA can refer to a mid-tracrRNA. A tracrRNA can refer to aminimum tracrRNA sequence.

CRISPR Systems

A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) canbe a genomic locus found in the genomes of many prokaryotes (e.g.,bacteria and archaea). CRISPR loci can provide resistance to foreigninvaders (e.g, virus, phage) in prokaryotes. In this way, the CRISPRsystem can be thought to function as a type of immune system to helpdefend prokaryotes against foreign invaders. There can be three stagesof CRISPR locus function: integration of new sequences into the locus,biogenesis of CRISPR RNA (crRNA), and silencing of foreign invadernucleic acid. There can be four types of CRISPR systems (e.g., Type I,Type II, Type III, TypeU).

A CRISPR locus can include a number of short repeating sequencesreferred to as “repeats.” Repeats can form hairpin structures and/orrepeats can be unstructured single-stranded sequences. The repeats canoccur in clusters. Repeats sequences can frequently diverge betweenspecies. Repeats can be regularly interspaced with unique interveningsequences referred to as “spacers,” resulting in a repeat-spacer-repeatlocus architecture. Spacers can be identical to or have high homologywith known foreign invader sequences. A spacer-repeat unit can encode acrisprRNA (crRNA). A crRNA can refer to the mature form of thespacer-repeat unit. A crRNA can comprise a “seed” sequence that can beinvolved in targeting a target nucleic acid (e.g., possibly as asurveillance mechansim against foreign nucleic acid). A seed sequencecan be located at the 5′ or 3′ end of the crRNA.

A CRISPR locus can comprise polynucleotide sequences encoding for CrisprAssociated Genes (Cas) genes. Cas genes can be involved in thebiogenesis and/or the interference stages of crRNA function. Cas genescan display extreme sequence (e.g., primary sequence) divergence betweenspecies and homologues. For example, Cas1 homologues can comprise lessthan 10% primary sequence identity between homologues. Some Cas genescan comprise homologous secondary and/or tertiary structures. Forexample, despite extreme sequence divergence, many members of the Cas6family of CRISPR proteins comprise a N-terminal ferredoxin-like fold.Cas genes can be named according to the organism from which they arederived. For example, Cas genes in Staphylococcus epidermidis can bereferred to as Csm-type, Cas genes in Streptococcus thermophilus can bereferred to as Csn-type, and Cas genes in Pyrococcus furiosus can bereferred to as Cmr-type.

Integration

The integration stage of CRISPR system can refer to the ability of theCRISPR locus to integrate new spacers into the crRNA array upon beinginfected by a foreign invader. Acquisition of the foreign invaderspacers can help confer immunity to subsequent attacks by the sameforeign invader. Integration can occur at the leader end of the CRISPRlocus. Cas proteins (e.g., Cas1 and Cas2) can be involved in integrationof new spacer sequences. Integration can proceed similarly for sometypes of CRISPR systems (e.g., Type I-III).

Biogenesis

Mature crRNAs can be processed from a longer polycistronic CRISPR locustranscript (i.e., pre-crRNA array). A pre-crRNA array can comprise aplurality of crRNAs. The repeats in the pre-crRNA array can berecognized by Cas genes. Cas genes can bind to the repeats and cleavethe repeats. This action can liberate the plurality of crRNAs. crRNAscan be subjected to further events to produce the mature crRNA form suchas trimming (e.g., with an exonuclese). A crRNA may comprise all, some,or none of the CRISPR repeat sequence.

Interference

Interference can refer to the stage in the CRISPR system that isfunctionally responsible for combating infection by a foreign invader.CRISPR interference can follow a similar mechanism to RNA interference(RNAi (e.g., wherein a target RNA is targeted (e.g., hybridzed) by ashort interfering RNA (siRNA)), which can result in target RNAdegradation and/or destabilization. CRISPR systems can performinterference of a target nucleic acid by coupling crRNAs and Cas genes,thereby forming CRISPR ribonucleoproteins (crRNPs). crRNA of the crRNPcan guide the crRNP to foreign invader nucleic acid, (e.g., byrecognizing the foreign invader nucleic acid through hybridization).Hybridized target foreign invader nucleic acid-crRNA units can besubjected to cleavage by Cas proteins. Target nucleic acid interferencemay require a spacer adjacent motif (PAM) in a target nucleic acid.

Types of CRISPR Systems

There can be four types of CRISPR systems: Type I, Type II, Type III,and Type U. More than one CRISPR type system can be found in anorganism. CRISPR systems can be complementary to each other, and/or canlend functional units in trans to facilitate CRISPR locus processing.

Type I CRISPR Systems

crRNA biogenesis in Type I CRISPR systems can comprise endoribonucleasecleavage of repeats in the pre-crRNA array, which can result in aplurality of crRNAs. crRNAs of Type I systems may not be subjected tocrRNA trimming. A crRNA can be processed from a pre-crRNA array by amulti-protein complex called Cascade (originating from CRISPR-associatedcomplex for antiviral defense). Cascade can comprise protein subunits(e.g, CasA-CasE). Some of the subunits can be members of the RepeatAssociated Mysterious Protein (RAMP) superfamily (e.g., Cas5 and Casiofamilies). The Cascade-crRNA complex (i.e., interference complex) canrecognize target nucleic acid through hybridization of the crRNA withthe target nucleic acid. The Cascade interference complex can recruitthe Cas3 helicase/nuclease which can act in trans to facilitate cleavageof target nucleic acid. The Cas3 nuclease can cleave target nucleic acid(e.g., with its HD nuclease domain). Target nucleic acid in a Type ICRISPR system can comprise a PAM. Target nucleic acid in a Type I CRISPRsystem can be DNA.

Type I systems can be further subdivided by their species of origin.Type I systems can comprise: Types IA (Aeropyrum pernix or CASS5); IB(Thermotoga neapolitana-Haloarcula marismortui or CASS7); IC(Desulfovibrio vulgaris or CASS1); ID; IE (Escherichia coli or CASS2);and IF (Yersinia pestis or CASS3) subfamilies.

Type II CRISPR Systems

crRNA biogenesis in a Type II CRISPR system can comprise atrans-activating CRISPR RNA (tracrRNA). A tracrRNA can be modified byendogenous RNaseIII. The tracrRNA of the complex can hybridize to acrRNA repeat in the pre-crRNA array. Endogenous RnaseIII can berecruited to cleave the pre-crRNA. Cleaved crRNAs can be subjected toexoribonuclease trimming to produce the mature crRNA form (e.g., 5′trimming). The tracrRNA can remain hybridized to the crRNA. The tracrRNAand the crRNA can associate with a site-directed polypeptide (e.g.,Cas9). The crRNA of the crRNA-tracrRNA-Cas9 complex can guide thecomplex to a target nucleic acid to which the crRNA can hybridize.Hybridization of the crRNA to the target nucleic acid can activate Cas9for target nucleic acid cleavage. Target nucleic acid in a Type IICRISPR system can comprise a PAM. In some embodiments, a PAM isessential to facilitate binding of a site-directed polypeptide (e.g.,Cas9) to a target nucleic acid. Type II systems can be furthersubdivided into II-A (Nmeni or CASS4) and II-B (Nmeni or CASS4).

Type III CRISPR Systems

crRNA biogenesis in Type III CRISPR systems can comprise a step ofendoribonuclease cleavage of repeats in the pre-crRNA array, which canresult in a plurality of crRNAs. Repeats in the Type III CRISPR systemcan be unstructured single-stranded regions. Repeats can be recognizedand cleaved by a member of the RAMP superfamily of endoribonucleases(e.g., Cas6). crRNAs of Type III (e.g., Type III-B) systems may besubjected to crRNA trimming (e.g., 3′ trimming). Type III systems cancomprise a polymerase-like protein (e.g., Cas10). Cas10 can comprise adomain homologous to a palm domain.

Type III systems can process pre-crRNA with a complex comprising aplurality of RAMP superfamily member proteins and one or more CRISPRpolymerase-like proteins. Type III systems can be divided into III-A andIII-B. An interference complex of the Type III-A system (i.e., Csmcomplex) can target plasmid nucleic acid. Cleavage of the plasmidnucleic acid can occur with the HD nuclease domain of a polymerase-likeprotein in the complex. An interference complex of the Type III-B system(i.e., Cmr complex) can target RNA.

Type U CRISPR Systems

Type U CRISPR systems may not comprise the signature genes of either ofthe Type I-III CRISPR systems (e.g., Cas3, Cas9, Cas6, Cas1, Cas2).Examples of Type U CRISPR Cas genes can include, but are not limited to,Csf1, Csf2, Csf3, Csf4. Type U Cas genes may be very distant homologuesof Type I-III Cas genes. For example, Csf3 may be highly diverged butfunctionally similar to Cas5 family members. A Type U system mayfunction complementarily in trans with a Type I-III system. In someinstances, Type U systems may not be associated with processing CRISPRarrays. Type U systems may represent an alternative foreign invaderdefense system.

RAMP Superfamily

Repeat Associated Mysterious Proteins (RAMP proteins) can becharacterized by a protein fold comprising a βαββαβ[beta-alpha-beta-beta-alpha-beta] motif of β-strands (β) and α-helices(α). A RAMP protein can comprise an RNA recognition motif (RRM) (whichcan comprise a ferredoxin or ferredoxin-like fold). RAMP proteins cancomprise an N-terminal RRM. The C-terminal domain of RAMP proteins canvary, but can also comprise an RRM. RAMP family members can recognizestructured and/or unstructured nucleic acid. RAMP family members canrecognize single-stranded and/or double-stranded nucleic acid. RAMPproteins can be involved in the biogenesis and/or the interference stageof CRISPR Type I and Type III systems. RAMP superfamily members cancomprise members of the Cas7, Cas6, and Cas5 families. RAMP superfamilymembers can be endoribonucleases.

RRM domains in the RAMP superfamily can be extremely divergent. RRMdomains can comprise at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or 100% sequence orstructural homology to a wild type exemplary RRM domain (e.g., an RRMdomain from Cas7). RRM domains can comprise at most about 5%, at mostabout 10%, at most about 15%, at most about 20%, at most about 25%, atmost about 30%, at most about 35%, at most about 40%, at most about 45%,at most about 50%, at most about 55%, at most about 60%, at most about65%, at most about 70%, at most about 75%, at most about 80%, at mostabout 85%, at most about 90%, at most about 95%, or 100% sequence orstructural homology to a wild type exemplary RRM domain (e.g., an RRMdomain from Cas7).

Cas7 Family

Cas7 family members can be a subclass of RAMP family proteins. Cas7family proteins can be categorized in Type I CRISPR systems. Cas7 familymembers may not comprise a glycine rich loop that is familiar to someRAMP family members. Cas7 family members can comprise one RRM domain.Cas7 family members can include, but are not limited to, Cas7 (COG1857),Cas7 (COG3649), Cas7 (CT1975), Csy3, Csm3, Cmr6, Csm5, Cmr4, Cmr1, Csf2,and Csc2.

Cas6 Family

The Cas6 family can be a RAMP subfamily. Cas6 family members cancomprise two RNA recognition motif (RRM)-like domains. A Cas6 familymember (e.g., Cas6f) can comprise a N-terminal RRM domain and a distinctC-terminal domain that may show weak sequence similarity or structuralhomology to an RRM domain. Cas6 family members can comprise a catalytichistidine that may be involved in endoribonuclease activity. Acomparable motif can be found in Cas5 and Cas7 RAMP families. Cas6family members can include, but are not limited to, Cas6, Cas6e, Cas6f(e.g., Csy4).

Cas5 Family

The Cas5 family can be a RAMP subfamily. The Cas5 family can be dividedinto two subgroups: one subgroup that can comprise two RRM domains, andone subgroup that can comprise one RRM domain. Cas5 family members caninclude, but are not limited to, Csm4, Csx10, Cmr3, Cas5, Cas5(BH0337),Csy2, Csc1, Csf3.

Cas Genes

Exemplary CRISPR Cas genes can include Cas1, Cas2, Cas3′ (Cas3-prime),Cas3″ (Cas3-double prime), Cas4, Cas5, Cas6, Cas6e (formerly referred toas CasE, Cse3), Cas6f (i.e., Csy4), Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c,Cas9, Cas10, Cas10d, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5,Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1,Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1,Csf2, Csf3, Csf4. Table 1 provides an exemplary categorization of CRISPRCas genes by CRISPR system type.

The CRISPR-Cas gene naming system has undergone extensive rewritingsince the Cas genes were discovered. For the purposes of thisapplication, Cas gene names used herein are based on the naming systemoutlined in Makarova et al. Evolution and classification of theCRISPR-Cas systems. Nature Reviews Microbiology. 2011 June; 9(6):467-477. Doi:10.1038/nrmicro2577.

TABLE 1 Exemplary classification of CRISPR Cas genes by CRISPR TypeSystem type or subtype Gene Name Type I cas1, cas2, cas3′ Type II cas1,cas2, cas9 Type III cas1, cas2, cas10 Subtype I-A cas3″, cas4, cas5,cas6, cas7, cas8a1, cas8a2, csa5 Subtype I-B cas3″, cas4, cas5, cas6,cas7, cas8b Subtype I-C cas4, cas5, cas7, cas8c Subtype I-D cas4, cas6,cas10d, csc1, csc2 Subtype I-E cas5, cas6e, cas7, cse1, cse2 Subtype I-Fcas6f, csy1, csy2, csy3 Subtype II-A csn2 Subtype II-B cas4 SubtypeIII-A cas6, csm2, csm3, csm4, csm5, csm6 Subtype III-B cas6, cmr1, cmr3,cmr4, cmr5, cmr6 Subtype I-U csb1, csb2, csb3, csx17, csx14, csx10Subtype III-U csx16, csaX, csx3, csx1 Unknown csx15 Type U csf1, csf2,csf3, csf4

Site-Directed Polypeptides

A site-directed polypeptide can be a polypeptide that can bind to atarget nucleic acid. A site-directed polypeptide can be a nuclease.

A site-directed polypeptide can comprise a nucleic acid-binding domain.The nucleic acid-binding domain can comprise a region that contacts anucleic acid. A nucleic acid-binding domain can comprise a nucleic acid.A nucleic acid-binding domain can comprise a proteinaceous material. Anucleic acid-binding domain can comprise nucleic acid and aproteinaceous material. A nucleic acid-binding domain can comprise RNA.There can be a single nucleic acid-binding domain. Examples of nucleicacid-binding domains can include, but are not limited to, ahelix-turn-helix domain, a zinc finger domain, a leucine zipper (bZIP)domain, a winged helix domain, a winged helix turn helix domain, ahelix-loop-helix domain, a HMG-box domain, a Wor3 domain, animmunoglobulin domain, a B3 domain, a TALE domain, a RNA-recognitionmotif domain, a double-stranded RNA-binding motif domain, adouble-stranded nucleic acid binding domain, a single-stranded nucleicacid binding domains, a KH domain, a PUF domain, a RGG box domain, aDEAD/DEAH box domain, a PAZ domain, a Piwi domain, and a cold-shockdomain.

A nucleic acid-binding domain can be a domain of an argonaute protein.An argonaute protein can be a eukaryotic argonaute or a prokaryoticargonaute. An argonaute protein can bind RNA, DNA, or both RNA and DNA.An argonaute protein can cleaved RNA, or DNA, or both RNA and DNA. Insome instances, an argonaute protein binds a DNA and cleaves a targetDNA.

In some instances, two or more nucleic acid-binding domains can belinked together. Linking a plurality of nucleic acid-binding domainstogether can provide increased polynucleotide targeting specificity. Twoor more nucleic acid-binding domains can be linked via one or morelinkers. The linker can be a flexible linker. Linkers can comprise 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40 or more amino acids in length. Linkers cancomprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% glycine content. Linkerscan comprise at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% glycine content.Linkers can comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% serinecontent. Linkers can comprise at most 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%serine content.

Nucleic acid-binding domains can bind to nucleic acid sequences. Nucleicacid binding domains can bind to nucleic acids through hybridization.Nucleic acid-binding domains can be engineered (e.g. engineered tohybridize to a sequence in a genome). A nucleic acid-binding domain canbe engineered by molecular cloning techniques (e.g., directed evolution,site-specific mutation, and rational mutagenesis).

A site-directed polypeptide can comprise a nucleic acid-cleaving domain.The nucleic acid-cleaving domain can be a nucleic acid-cleaving domainfrom any nucleic acid-cleaving protein. The nucleic acid-cleaving domaincan originate from a nuclease. Suitable nucleic acid-cleaving domainsinclude the nucleic acid-cleaving domain of endonucleases (e.g., APendonuclease, RecBCD enonuclease, T7 endonuclease, T4 endonuclease IV,Bal 31 endonuclease, Endonucleasel (endo I), Micrococcal nuclease,Endonuclease II (endo VI, exo III)), exonucleases, restrictionnucleases, endoribonucleases, exoribonucleases, RNases (e.g., RNAse I,II, or III). In some instances, the nucleic acid-cleaving domain canoriginate from the FokI endonuclease. A site-directed polypeptide cancomprise a plurality of nucleic acid-cleaving domains. Nucleicacid-cleaving domains can be linked together. Two or more nucleicacid-cleaving domains can be linked via a linker. In some embodiments,the linker can be a flexible linker. Linkers can comprise 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 30, 35, 40 or more amino acids in length. In some embodiments, asite-directed polypeptide can comprise the plurality of nucleicacid-cleaving domains.

A site-directed polypeptide (e.g., Cas9, argonaute) can comprise two ormore nuclease domains. Cas9 can comprise a HNH or FINE-like nucleasedomain and/or a RuvC or RuvC-like nuclease domain. HNH or FINE-likedomains can comprise a McrA-like fold. HNH or FINE-like domains cancomprise two antiparallel β-strands and an α-helix. HNH or FINE-likedomains can comprise a metal binding site (e.g., divalent cation bindingsite). HNH or HNH-like domains can cleave one strand of a target nucleicacid (e.g., complementary strand of the crRNA targeted strand). Proteinsthat comprise an HNH or HNH-like domain can include endonucleases,clicins, restriction endonucleases, transposases, and DNA packagingfactors.

RuvC or RuvC-like domains can comprise an RNaseH or RNaseH-like fold.RuvC/RNaseH domains can be involved in a diverse set of nucleicacid-based functions including acting on both RNA and DNA. The RNaseHdomain can comprise 5 β-strands surrounded by a plurality of α-helices.RuvC/RNaseH or RuvC/RNaseH-like domains can comprise a metal bindingsite (e.g., divalent cation binding site). RuvC/RNaseH orRuvC/RNaseH-like domains can cleave one strand of a target nucleic acid(e.g., non-complementary strand of the crRNA targeted strand). Proteinsthat comprise a RuvC, RuvC-like, or RNaseH-like domain can includeRNaseH, RuvC, DNA transposases, retroviral integrases, and Argonautproteins).

The site-directed polypeptide can be an endoribonuclease. Thesite-directed polypeptide can be an enzymatically inactive site-directedpolypeptide. The site-directed polypeptide can be a conditionallyenzymatically inactive site-directed polypeptide.

Site-directed polypeptides can introduce double-stranded breaks orsingle-stranded breaks in nucleic acid, (e.g. genomic DNA). Thedouble-stranded break can stimulate a cell's endogenous DNA-repairpathways (e.g. homologous recombination and non-homologous end joining(NHEJ) or alternative non-homologues end joining (A-NHEJ)). NHEJ canrepair cleaved target nucleic acid without the need for a homologoustemplate. This can result in deletions of the target nucleic acid.Homologous recombination (HR) can occur with a homologous template. Thehomologous template can comprise sequences that are homologous tosequences flanking the target nucleic acid cleavage site. After a targetnucleic acid is cleaved by a site-directed polypeptide the site ofcleavage can be destroyed (e.g., the site may not be accessible foranother round of cleavage with the original nucleica acid-targetingnucleic acid and site-directed polypeptide).

In some cases, homologous recombination can insert an exogenouspolynucleotide sequence into the target nucleic acid cleavage site. Anexogenous polynucleotide sequence can be called a donor polynucleotide.In some instances of the methods of the disclosure the donorpolynucleotide, a portion of the donor polynucleotide, a copy of thedonor polynucleotide, or a portion of a copy of the donor polynucleotidecan be inserted into the target nucleic acid cleavage site. A donorpolynucleotide can be an exogenous polynucleotide sequence. A donorpolynucleotide can be a sequence that does not naturally occur at thetarget nucleic acid cleavage site. A vector can comprise a donorpolynucleotide. The modifications of the target DNA due to NHEJ and/orHR can lead to, for example, mutations, deletions, alterations,integrations, gene correction, gene replacement, gene tagging, transgeneinsertion, nucleotide deletion, gene disruption, and/or gene mutation.The process of integrating non-native nucleic acid into genomic DNA canbe referred to as genome engineering.

In some cases, the site-directed polypeptide can comprise an amino acidsequence having at most 10%, at most 15%, at most 20%, at most 30%, atmost 40%, at most 50%, at most 60%, at most 70%, at most 75%, at most80%, at most 85%, at most 90%, at most 95%, at most 99%, or 100%, aminoacid sequence identity to a wild type exemplary site-directedpolypeptide (e.g, Cas9 from S. pyogenes, SEQ ID NO: 8).

In some cases, the site-directed polypeptide can comprise an amino acidsequence having at least 10%, at least 15%, 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%,amino acid sequence identity to a wild type exemplary site-directedpolypeptide (e.g., Cas9 from S. pyogenes, SEQ ID NO: 8).

In some cases, the site-directed polypeptide can comprise an amino acidsequence having at most 10%, at most 15%, at most 20%, at most 30%, atmost 40%, at most 50%, at most 60%, at most 70%, at most 75%, at most80%, at most 85%, at most 90%, at most 95%, at most 99%, or 100%, aminoacid sequence identity to the nuclease domain of a wild type exemplarysite-directed polypeptide (e.g, Cas9 from S. pyogenes, SEQ ID NO: 8).

A site-directed polypeptide can comprise at least 70, 75, 80, 85, 90,95, 97, 99, or 100% identity to wild-type site-directed polypeptide(e.g., Cas9 from S. pyogenes, SEQ ID NO: 8) over 10 contiguous aminoacids. A site-directed polypeptide can comprise at most 70, 75, 80, 85,90, 95, 97, 99, or 100% identity to wild-type site-directed polypeptide(e.g., Cas9 from S. pyogenes, SEQ ID NO: 8) over 10 contiguous aminoacids. A site-directed polypeptide can comprise at least 70, 75, 80, 85,90, 95, 97, 99, or 100% identity to a wild-type site-directedpolypeptide (e.g., Cas9 from S. pyogenes, SEQ ID NO: 8) over 10contiguous amino acids in a HNH nuclease domain of the site-directedpolypeptide. A site-directed polypeptide can comprise at most 70, 75,80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directedpolypeptide (e.g., Cas9 from S. pyogenes, SEQ ID NO: 8) over 10contiguous amino acids in a HNH nuclease domain of the site-directedpolypeptide. A site-directed polypeptide can comprise at least 70, 75,80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directedpolypeptide (e.g., Cas9 from S. pyogenes, SEQ ID NO: 8) over 10contiguous amino acids in a RuvC nuclease domain of the site-directedpolypeptide. A site-directed polypeptide can comprise at most 70, 75,80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directedpolypeptide (e.g., Cas9 from S. pyogenes, SEQ ID NO: 8) over 10contiguous amino acids in a RuvC nuclease domain of the site-directedpolypeptide.

In some cases, the site-directed polypeptide can comprise an amino acidsequence having at least 10%, at least 15%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 99%, or100%, amino acid sequence identity to the nuclease domain of a wild typeexemplary site-directed polypeptide (e.g., Cas9 from S. pyogenes).

The site-directed polypeptide can comprise a modified form of a wildtype exemplary site-directed polypeptide. The modified form of the wildtype exemplary site-directed polypeptide can comprise an amino acidchange (e.g., deletion, insertion, or substitution) that reduces thenucleic acid-cleaving activity of the site-directed polypeptide. Forexample, the modified form of the wild type exemplary site-directedpolypeptide can have less than less than 90%, less than 80%, less than70%, less than 60%, less than 50%, less than 40%, less than 30%, lessthan 20%, less than 10%, less than 5%, or less than 1% of the nucleicacid-cleaving activity of the wild-type exemplary site-directedpolypeptide (e.g., Cas9 from S. pyogenes). The modified form of thesite-directed polypeptide can have no substantial nucleic acid-cleavingactivity. When a site-directed polypeptide is a modified form that hasno substantial nucleic acid-cleaving activity, it can be referred to as“enzymatically inactive.”

The modified form of the wild type exemplary site-directed polypeptidecan have more than 90%, more than 80%, more than 70%, more than 60%,more than 50%, more than 40%, more than 30%, more than 20%, more than10%, more than 5%, or more than 1% of the nucleic acid-cleaving activityof the wild-type exemplary site-directed polypeptide (e.g., Cas9 from S.pyogenes).

The modified form of the site-directed polypeptide can comprise amutation. The modified form of the site-directed polypeptide cancomprise a mutation such that it can induce a single stranded break(SSB) on a target nucleic acid (e.g., by cutting only one of thesugar-phosphate backbones of the target nucleic acid). The mutation canresult in less than 90%, less than 80%, less than 70%, less than 60%,less than 50%, less than 40%, less than 30%, less than 20%, less than10%, less than 5%, or less than 1% of the nucleic acid-cleaving activityin one or more of the plurality of nucleic acid-cleaving domains of thewild-type site directed polypeptide (e.g., Cas9 from S. pyogenes). Themutation can result in one or more of the plurality of nucleicacid-cleaving domains retaining the ability to cleave the complementarystrand of the target nucleic acid but reducing its ability to cleave thenon-complementary strand of the target nucleic acid. The mutation canresult in one or more of the plurality of nucleic acid-cleaving domainsretaining the ability to cleave the non-complementary strand of thetarget nucleic acid but reducing its ability to cleave the complementarystrand of the target nucleic acid. For example, residues in the wildtype exemplary S. pyogenes Cas9 polypeptide such as Asp10, His840,Asn854 and Asn856 can be mutated to inactivate one or more of theplurality of nucleic acid-cleaving domains (e.g., nuclease domains). Theresidues to be mutated can correspond to residues Asp10, His840, Asn854and Asn856 in the wild type exemplary S. pyogenes Cas9 polypeptide(e.g., as determined by sequence and/or structural alignment).Non-limiting examples of mutations can include D10A, H840A, N854A orN856A. One skilled in the art will recognize that mutations other thanalanine substitutions are suitable.

A D10A mutation can be combined with one or more of H840A, N854A, orN856A mutations to produce a site-directed polypeptide substantiallylacking DNA cleavage activity. A H840A mutation can be combined with oneor more of D10A, N854A, or N856A mutations to produce a site-directedpolypeptide substantially lacking DNA cleavage activity. A N854Amutation can be combined with one or more of H840A, D10A, or N856Amutations to produce a site-directed polypeptide substantially lackingDNA cleavage activity. A N856A mutation can be combined with one or moreof H840A, N854A, or D10A mutations to produce a site-directedpolypeptide substantially lacking DNA cleavage activity. Site-directedpolypeptides that comprise one substantially inactive nuclease domaincan be referred to as nickases.

Mutations of the disclosure can be produced by site-directed mutation.Mutations can include substitutions, additions, and deletions, or anycombination thereof. In some instances, the mutation converts themutated amino acid to alanine. In some instances, the mutation convertsthe mutated amino acid to another amino acid (e.g., glycine, serine,threonine, cysteine, valine, leucine, isoleucine, methionine, proline,phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid,asparagines, glutamine, histidine, lysine, or arginine). The mutationcan convert the mutated amino acid to a non-natural amino acid (e.g.,selenomethionine). The mutation can convert the mutated amino acid toamino acid mimics (e.g., phosphomimics). The mutation can be aconservative mutation. For example, the mutation can convert the mutatedamino acid to amino acids that resemble the size, shape, charge,polarity, conformation, and/or rotamers of the mutated amino acids(e.g., cysteine/serine mutation, lysine/asparagine mutation,histidine/phenylalanine mutation).

In some instances, the site-directed polypeptide (e.g., variant,mutated, enzymatically inactive and/or conditionally enzymaticallyinactive site-directed polypeptide) can target nucleic acid. Thesite-directed polypeptide (e.g., variant, mutated, enzymaticallyinactive and/or conditionally enzymatically inactive endoribonuclease)can target RNA. Site-directed polypeptides that can target RNA caninclude members of other CRISPR subfamilies such as Cas6 and Cas5.

The site-directed polypeptide can comprise one or more non-nativesequences (e.g., a fusion).

A site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), a nucleic acid binding domain, and two nucleic acidcleaving domains (i.e., an HNH domain and a RuvC domain).

A site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., an HNHdomain and a RuvC domain).

A site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains, wherein oneor both of the nucleic acid cleaving domains comprise at least 50% aminoacid identity to a nuclease domain from Cas9 from a bacterium (e.g., S.pyogenes).

A site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., an HNHdomain and a RuvC domain), and a linker linking the site-directedpolypeptide to a non-native sequence.

A site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., an HNHdomain and a RuvC domain), wherein the site-directed polypeptidecomprises a mutation in one or both of the nucleic acid cleaving domainsthat reduces the cleaving activity of the nuclease domains by at least50%.

A site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., an HNHdomain and a RuvC domain), wherein one of the nuclease domains comprisesmutation of aspartic acid 10, and/or wherein one of the nuclease domainscomprises mutation of histidine 840, and wherein the mutation reduce thecleaving activity of the nuclease domains by at least 50%.

Endoribonucleases

In some embodiments, a site-directed polypeptide can be anendoribonuclease.

In some cases, the endoribonuclease can comprise an amino acid sequencehaving at most about 20%, at most about 30%, at most about 40%, at mostabout 50%, at most about 60%, at most about 70%, at most about 75%, atmost about 80%, at most about 85%, at most about 90%, at most about 95%,at most about 99%, or 100%, amino acid sequence identity and/or homologyto a wild type reference endoribonuclease. The endoribonuclease cancomprise an amino acid sequence having at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 99%,or 100%, amino acid sequence identity and/or homology to a wild typereference endoribonuclease (e.g., Csy4 from P. aeruginosa). Thereference endoribonuclease can be a Cas6 family member (e.g., Csy4,Cas6). The reference endoribonuclease can be a Cas5 family member (e.g.,Cas5 from D. vulgaris). The reference endoribonuclease can be a Type ICRISPR family member (e.g., Cas3). The reference endoribonucleases canbe a Type II family member. The reference endoribonuclease can be a TypeIII family member (e.g., Cas6). A reference endoribonuclease can be amember of the Repeat Associated Mysterious Protein (RAMP) superfamily(e.g., Cas7).

The endoribonuclease can comprise amino acid modifications (e.g.,substitutions, deletions, additions etc). The endoribonuclease cancomprise one or more non-native sequences (e.g., a fusion, an affinitytag). The amino acid modifications may not substantially alter theactivity of the endoribonuclease. An endoribonuclease comprising aminoacid modifications and/or fusions can retain at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 97% or 100% activity of the wild-typeendoribonuclease.

The modification can result alteration of the enzymatic activity of theendoribonuclease. The modification can result in less than 90%, lessthan 80%, less than 70%, less than 60%, less than 50%, less than 40%,less than 30%, less than 20%, less than 10%, less than 5%, or less than1% of the endoribonuclease. In some instances, the modification occursin the nuclease domain of an endoribonuclease. Such modifications canresult in less than 90%, less than 80%, less than 70%, less than 60%,less than 50%, less than 40%, less than 30%, less than 20%, less than10%, less than 5%, or less than 1% of the nucleic acid-cleaving abilityin one or more of the plurality of nucleic acid-cleaving domains of thewild-type endoribonuclease.

Conditionally Enzymatically Inactive Endoribonucleases

In some embodiments, an endoribonuclease can be conditionallyenzymatically inactive. A conditionally enzymatically inactiveendoribonuclease can bind to a polynucleotide in a sequence-specificmanner. A conditionally enzymatically inactive endoribonuclease can binda polynucleotide in a sequence-specific manner, but cannot cleave thetarget polyribonucleotide.

In some cases, the conditionally enzymatically inactive endoribonucleasecan comprise an amino acid sequence having up to about 20%, up to about30%, up to about 40%, up to about 50%, up to about 60%, up to about 70%,up to about 75%, up to about 80%, up to about 85%, up to about 90%, upto about 95%, up to about 99%, or 100%, amino acid sequence identityand/or homology to a reference conditionally enzymaticallyendoribonuclease (e.g., Csy4 from P. aeruginosa). In some cases, theconditionally enzymatically inactive endoribonuclease can comprise anamino acid sequence having at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 99%, or 100%, aminoacid sequence identity and/or homology to a reference conditionallyenzymatically endoribonuclease (e.g., Csy4 from P. aeruginosa).

The conditionally enzymatically inactive endoribonuclease can comprise amodified form of an endoribonuclease. The modified form of theendoribonuclease can comprise an amino acid change (e.g., deletion,insertion, or substitution) that reduces the nucleic acid-cleavingactivity of the endoribonuclease. For example, the modified form of theconditionally enzymatically inactive endoribonuclease can have less thanless than 90%, less than 80%, less than 70%, less than 60%, less than50%, less than 40%, less than 30%, less than 20%, less than 10%, lessthan 5%, or less than 1% of the nucleic acid-cleaving activity of thereference (e.g., wild-type) conditionally enzymatically inactiveendoribonuclease (e.g., Csy4 from P. aeruginosa). The modified form ofthe conditionally enzymatically inactive endoribonuclease can have nosubstantial nucleic acid-cleaving activity. When a conditionallyenzymatically inactive endoribonuclease is a modified form that has nosubstantial nucleic acid-cleaving activity, it can be referred to as“enzymatically inactive.”

The modified form of the conditionally enzymatically inactiveendoribonuclease can comprise a mutation that can result in reducednucleic acid-cleaving ability (i.e., such that the conditionallyenzymatically inactive endoribonuclease can be enzymatically inactive inone or more of the nucleic acid-cleaving domains). The mutation canresult in less than 90%, less than 80%, less than 70%, less than 60%,less than 50%, less than 40%, less than 30%, less than 20%, less than10%, less than 5%, or less than 1% of the nucleic acid-cleaving abilityin one or more of the plurality of nucleic acid-cleaving domains of thewild-type endoribonuclease (e.g., Csy4 from P. aeruginosa). The mutationcan occur in the nuclease domain of the endoribonuclease. The mutationcan occur in a ferredoxin-like fold. The mutation can comprise themutation of a conserved aromatic amino acid. The mutation can comprisethe mutation of a catalytic amino acid. The mutation can comprise themutation of a histidine. For example, the mutation can comprise a H29Amutation in Csy4 (e.g., Csy4 from P. aeruginosa), or any correspondingresidue to H29A as determined by sequence and/or structural alignment.Other residues can be mutated to achieve the same effect (i.e.inactivate one or more of the plurality of nuclease domains).

Mutations of the invention can be produced by site-directed mutation.Mutations can include substitutions, additions, and deletions, or anycombination thereof. In some instances, the mutation converts themutated amino acid to alanine. In some instances, the mutation convertsthe mutated amino acid to another amino acid (e.g., glycine, serine,threonine, cysteine, valine, leucine, isoleucine, methionine, proline,phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid,asparagines, glutamine, histidine, lysine, or arginine). The mutationcan convert the mutated amino acid to a non-natural amino acid (e.g.,selenomethionine). The mutation can convert the mutated amino acid toamino acid mimics (e.g., phosphomimics). The mutation can be aconservative mutation. For example, the mutation can convert the mutatedamino acid to amino acids that resemble the size, shape, charge,polarity, conformation, and/or rotamers of the mutated amino acids(e.g., cysteine/serine mutation, lysine/asparagine mutation,histidine/phenylalanine mutation).

A conditionally enzymatically inactive endoribonuclease can beenzymatically inactive in the absence of a reactivation agent (e.g.,imidazole). A reactivation agent can be an agent that mimics a histidineresidue (e.g., may have an imidazole ring). A conditionallyenzymatically inactive endoribonuclease can be activated by contact witha reactivation agent. The reactivation agent can comprise imidazole. Forexample, the conditionally enzymatically inactive endoribonuclease canbe enzymatically activated by contacting the conditionally enzymaticallyinactive endoribonuclease with imidazole at a concentration of fromabout 100 mM to about 500 mM. The imidazole can be at a concentration ofabout 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM,about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, orabout 600 mM. The presence of imidazole (e.g., in a concentration rangeof from about 100 mM to about 500 mM) can reactivate the conditionallyenzymatically inactive endoribonuclease such that the conditionallyenzymatically inactive endoribonuclease becomes enzymatically active,e.g., the conditionally enzymatically inactive endoribonuclease exhibitsat least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, or more than 95%, ofthe nucleic acid cleaving ability of a reference conditionallyenzymatically inactive endoribonuclease (e.g., Csy4 from P. aeruginosacomprising H29A mutation).

A conditionally enzymatically inactive endoribonuclease can comprise atleast 20% amino acid identity to Csy4 from P. aeruginosa, a mutation ofhistidine 29, wherein the mutation results in at least 50% reduction ofnuclease activity of the endoribonuclease, and wherein at least 50% ofthe lost nuclease activity can be restored by incubation of theendoribonuclease with at least 100 mM imidazole.

Codon-Optimization

A polynucleotide encoding a site-directed polypeptide and/or anendoribonuclease can be codon-optimized. This type of optimization canentail the mutation of foreign-derived (e.g., recombinant) DNA to mimicthe codon preferences of the intended host organism or cell whileencoding the same protein. Thus, the codons can be changed, but theencoded protein remains unchanged. For example, if the intended targetcell was a human cell, a human codon-optimized polynucleotide Cas9 couldbe used for producing a suitable site-directed polypeptide. As anothernon-limiting example, if the intended host cell were a mouse cell, thena mouse codon-optimized polynucleotide encoding Cas9 could be a suitablesite-directed polypeptide. A polynucleotide encoding a site-directedpolypeptide can be codon optimized for many host cells of interest. Ahost cell can be a cell from any organism (e.g. a bacterial cell, anarchaeal cell, a cell of a single-cell eukaryotic organism, a plantcell, an algal cell, e.g., Botryococcus braunii, Chlamydomonasreinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassumpatens C. Agardh, and the like, a fungal cell (e.g., a yeast cell), ananimal cell, a cell from an invertebrate animal (e.g. fruit fly,cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal(e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal(e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, anon-human primate, a human, etc.), etc. Codon optimization may not berequired. In some instances, codon optimization can be preferable.

Nucleic Acid-Targeting Nucleic Acid

The present disclosure provides for a nucleic acid-targeting nucleicacid that can direct the activities of an associated polypeptide (e.g.,a site-directed polypeptide) to a specific target sequence within atarget nucleic acid. The nucleic acid-targeting nucleic acid cancomprise nucleotides. The nucleic acid-targeting nucleic acid can beRNA. A nucleic acid-targeting nucleic acid can comprise a single guidenucleic acid-targeting nucleic acid. An exemplary single guide nucleicacid is depicted in FIG. 1A. The spacer extension 105 and the tracrRNAextension 135 can comprise elements that can contribute additionalfunctionality (e.g., stability) to the nucleic acid-targeting nucleicacid. In some embodiments the spacer extension 105 and the tracrRNAextension 135 are optional. A spacer sequence 110 can comprise asequence that can hybridize to a target nucleic acid sequence. Thespacer sequence 110 can be a variable portion of the nucleicacid-targeting nucleic acid. The sequence of the spacer sequence 110 canbe engineered to hybridize to the target nucleic acid sequence. TheCRISPR repeat 115 (i.e. referred to in this exemplary embodiment as aminimum CRISPR repeat) can comprise nucleotides that can hybridize to atracrRNA sequence 125 (i.e. referred to in this exemplary embodiment asa minimum tracrRNA sequence). The minimum CRISPR repeat 115 and theminimum tracrRNA sequence 125 can interact, the interacting moleculescomprising a base-paired, double-stranded structure. Together, theminimum CRISPR repeat 115 and the minimum tracrRNA sequence 125 canfacilitate binding to the site-directed polypeptide. The minimum CRISPRrepeat 115 and the minimum tracrRNA sequence 125 can be linked togetherto form a hairpin structure through the single guide connector 120. The3′ tracrRNA sequence 130 can comprise a protospacer adjacent motifrecognition sequence. The 3′ tracrRNA sequence 130 can be identical orsimilar to part of a tracrRNA sequence. In some embodiments, the 3′tracrRNA sequence 130 can comprise one or more hairpins.

In some embodiments, a nucleic acid-targeting nucleic acid can comprisea single guide nucleic acid-targeting nucleic acid as depicted in FIG.1B. A nucleic acid-targeting nucleic acid can comprise a spacer sequence140. A spacer sequence 140 can comprise a sequence that can hybridize tothe target nucleic acid sequence. The spacer sequence 140 can be avariable portion of the nucleic acid-targeting nucleic acid. The spacersequence 140 can be 5′ of a first duplex 145. The first duplex 145comprises a region of hybridization between a minimum CRISPR repeat 146and minimum tracrRNA sequence 147. The first duplex 145 can beinterrupted by a bulge 150. The bulge150 can comprise unpairednucleotides. The bulge 150 can be facilitate the recruitment of asite-directed polypeptide to the nucleic acid-targeting nucleic acid.The bulge 150 can be followed by a first stem 155. The first stem 155comprises a linker sequence linking the minimum CRISPR repeat 146 andthe minimum tracrRNA sequence 147. The last paired nucleotide at the 3′end of the first duplex 145 can be connected to a second linker sequence160. The second linker 160 can comprise a P-domain. The second linker160 can link the first duplex 145 to a mid-tracrRNA 165. Themid-tracrRNA 165 can, in some embodiments, comprise one or more hairpinregions. For example the mid-tracrRNA 165 can comprise a second stem 170and a third stem 175.

In some embodiments, the nucleic acid-targeting nucleic acid cancomprise a double guide nucleic acid structure. FIG. 2 depicts anexemplary double guide nucleic acid-targeting nucleic acid structure.Similar to the single guide nucleic acid structure of FIG. 1, the doubleguide nucleic acid structure can comprise a spacer extension 205, aspacer 210, a minimum CRISPR repeat 215, a minimum tracrRNA sequence230, a 3′ tracrRNA sequence 235, and a tracrRNA extension 240. However,a double guide nucleic acid-targeting nucleic acid may not comprise thesingle guide connector 120. Instead the minimum CRISPR repeat sequence215 can comprise a 3′ CRISPR repeat sequence 220 which can be similar oridentical to part of a CRISPR repeat. Similarly, the minimum tracrRNAsequence 230 can comprise a 5′ tracrRNA sequence 225 which can besimilar or identical to part of a tracrRNA. The double guide RNAs canhybridize together via the minimum CRISPR repeat 215 and the minimumtracrRNA sequence 230.

In some embodiments, the first segment (i.e., nucleic acid-targetingsegment) can comprise the spacer extension (e.g., 105/205) and thespacer (e.g., 110/210). The nucleic acid-targeting nucleic acid canguide the bound polypeptide to a specific nucleotide sequence withintarget nucleic acid via the above mentioned nucleic acid-targetingsegment.

In some embodiments, the second segment (i.e., protein binding segment)can comprise the minimum CRISPR repeat (e.g., 115/215), the minimumtracrRNA sequence (e.g., 125/230), the 3′ tracrRNA sequence (e.g.,130/235), and/or the tracrRNA extension sequence (e.g., 135/240). Theprotein-binding segment of a nucleic acid-targeting nucleic acid caninteract with a site-directed polypeptide. The protein-binding segmentof a nucleic acid-targeting nucleic acid can comprise two stretches ofnucleotides that that can hybridize to one another. The nucleotides ofthe protein-binding segment can hybridize to form a double-strandednucleic acid duplex. The double-stranded nucleic acid duplex can be RNA.The double-stranded nucleic acid duplex can be DNA.

In some instances, a nucleic acid-targeting nucleic acid can comprise,in the order of 5′ to 3′, a spacer extension, a spacer, a minimum CRISPRrepeat, a single guide connector, a minimum tracrRNA, a 3′ tracrRNAsequence, and a tracrRNA extension. In some instances, a nucleicacid-targeting nucleic acid can comprise, a tracrRNA extension, a3′tracrRNA sequence, a minimum tracrRNA, a single guide connector, aminimum CRISPR repeat, a spacer, and a spacer extension in any order.

A nucleic acid-targeting nucleic acid and a site-directed polypeptidecan form a complex. The nucleic acid-targeting nucleic acid can providetarget specificity to the complex by comprising a nucleotide sequencethat can hybridize to a sequence of a target nucleic acid. In otherwords, the site-directed polypeptide can be guided to a nucleic acidsequence by virtue of its association with at least the protein-bindingsegment of the nucleic acid-targeting nucleic acid. The nucleicacid-targeting nucleic acid can direct the activity of a Cas9 protein.The nucleic acid-targeting nucleic acid can direct the activity of anenzymatically inactive Cas9 protein.

Methods of the disclosure can provide for a genetically modified cell. Agenetically modified cell can comprise an exogenous nucleicacid-targeting nucleic acid and/or an exogenous nucleic acid comprisinga nucleotide sequence encoding a nucleic acid-targeting nucleic acid.

Spacer Extension Sequence

A spacer extension sequence can provide stability and/or provide alocation for modifications of a nucleic acid-targeting nucleic acid. Aspacer extension sequence can have a length of from about 1 nucleotideto about 400 nucleotides. A spacer extension sequence can have a lengthof more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360,380, 40, 1000, 2000, 3000, 4000, 5000, 6000, or 7000 or morenucleotides. A spacer extension sequence can have a length of less than1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140,160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000,2000, 3000, 4000, 5000, 6000, 7000 or more nucleotides. A spacerextension sequence can be less than 10 nucleotides in length. A spacerextension sequence can be between 10 and 30 nucleotides in length. Aspacer extension sequence can be between 30-70 nucleotides in length.

The spacer extension sequence can comprise a moiety (e.g., a stabilitycontrol sequence, an endoribonuclease binding sequence, a ribozyme). Amoiety can influence the stability of a nucleic acid targeting RNA. Amoiety can be a transcriptional terminator segment (i.e., atranscription termination sequence). A moiety of a nucleicacid-targeting nucleic acid can have a total length of from about 10nucleotides to about 100 nucleotides, from about 10 nucleotides (nt) toabout 20 nt, from about 20 nt to about 30 nt, from about 30 nt to about40 nt, from about 40 nt to about 50 nt, from about 50 nt to about 60 nt,from about 60 nt to about 70 nt, from about 70 nt to about 80 nt, fromabout 80 nt to about 90 nt, or from about 90 nt to about 100 nt, fromabout 15 nucleotides (nt) to about 80 nt, from about 15 nt to about 50nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt orfrom about 15 nt to about 25 nt. The moiety can be one that can functionin a eukaryotic cell. In some cases, the moiety can be one that canfunction in a prokaryotic cell. The moiety can be one that can functionin both a eukaryotic cell and a prokaryotic cell.

Non-limiting examples of suitable moieties can include: 5′ cap (e.g., a7-methylguanylate cap (m7 G)), a riboswitch sequence (e.g., to allow forregulated stability and/or regulated accessibility by proteins andprotein complexes), a sequence that forms a dsRNA duplex (i.e., ahairpin), a sequence that targets the RNA to a subcellular location(e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), a modification or sequence that provides abinding site for proteins (e.g., proteins that act on DNA, includingtranscriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like) a modification or sequence thatprovides for increased, decreased, and/or controllable stability, or anycombination thereof. A spacer extension sequence can comprise a primerbinding site, a molecular index (e.g., barcode sequence). The spacerextension sequence can comprise a nucleic acid affinity tag.

Spacer

The nucleic acid-targeting segment of a nucleic acid-targeting nucleicacid can comprise a nucleotide sequence (e.g., a spacer) that canhybridize to a sequence in a target nucleic acid. The spacer of anucleic acid-targeting nucleic acid can interact with a target nucleicacid in a sequence-specific manner via hybridization (i.e., basepairing). As such, the nucleotide sequence of the spacer may vary andcan determine the location within the target nucleic acid that thenucleic acid-targeting nucleic acid and the target nucleic acid caninteract.

The spacer sequence can hybridize to a target nucleic acid that islocated 5′ of spacer adjacent motif (PAM). Different organisms maycomprise different PAM sequences. For example, in S. pyogenes, the PAMcan be a sequence in the target nucleic acid that comprises the sequence5′-XRR-3′, where R can be either A or G, where X is any nucleotide and Xis immediately 3′ of the target nucleic acid sequence targeted by thespacer sequence.

The target nucleic acid sequence can be 20 nucleotides. The targetnucleic acid can be less than 20 nucleotides. The target nucleic acidcan be at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 ormore nucleotides. The target nucleic acid can be at most 5, 10, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. The targetnucleic acid sequence can be 20 bases immediately 5′ of the firstnucleotide of the PAM. For example, in a sequence comprising 5′-XRR-3′,the target nucleic acid can be the sequence that corresponds to the N's,wherein N is any nucleotide.

The nucleic acid-targeting sequence of the spacer that can hybridize tothe target nucleic acid can have a length at least about 6 nt. Forexample, the spacer sequence that can hybridize the target nucleic acidcan have a length at least about 6 nt, at least about 10 nt, at leastabout 15 nt, at least about 18 nt, at least about 19 nt, at least about20 nt, at least about 25 nt, at least about 30 nt, at least about 35 ntor at least about 40 nt, from about 6 nt to about 80 nt, from about 6 ntto about 50 nt, from about 6 nt to about 45 nt, from about 6 nt to about40 nt, from about 6 nt to about 35 nt, from about 6 nt to about 30 nt,from about 6 nt to about 25 nt, from about 6 nt to about 20 nt, fromabout 6 nt to about 19 nt, from about 10 nt to about 50 nt, from about10 nt to about 45 nt, from about 10 nt to about 40 nt, from about 10 ntto about 35 nt, from about 10 nt to about 30 nt, from about 10 nt toabout 25 nt, from about 10 nt to about 20 nt, from about 10 nt to about19 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt,from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, fromabout 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 ntto about 30 nt, from about 20 nt to about 35 nt, from about 20 nt toabout 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about50 nt, or from about 20 nt to about 60 nt. In some cases, the spacersequence that can hybridize the target nucleic acid can be 20nucleotides in length. The spacer that can hybridize the target nucleicacid can be 19 nucleotides in length.

The percent complementarity between the spacer sequence the targetnucleic acid can be at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 97%, at least about 98%,at least about 99%, or 100%. The percent complementarity between thespacer sequence the target nucleic acid can be at most about 30%, atmost about 40%, at most about 50%, at most about 60%, at most about 65%,at most about 70%, at most about 75%, at most about 80%, at most about85%, at most about 90%, at most about 95%, at most about 97%, at mostabout 98%, at most about 99%, or 100%. In some cases, the percentcomplementarity between the spacer sequence and the target nucleic acidcan be 100% over the six contiguous 5′-most nucleotides of the targetsequence of the complementary strand of the target nucleic acid. In somecases, the percent complementarity between the spacer sequence and thetarget nucleic acid can be at least 60% over about 20 contiguousnucleotides. In some cases, the percent complementarity between thespacer sequence and the target nucleic acid can be 100% over thefourteen contiguous 5′-most nucleotides of the target sequence of thecomplementary strand of the target nucleic acid and as low as 0% overthe remainder. In such a case, the spacer sequence can be considered tobe 14 nucleotides in length. In some cases, the percent complementaritybetween the spacer sequence and the target nucleic acid can be 100% overthe six contiguous 5′-most nucleotides of the target sequence of thecomplementary strand of the target nucleic acid and as low as 0% overthe remainder. In such a case, the spacer sequence can be considered tobe 6 nucleotides in length. The target nucleic acid can be more thanabout 50%, 60%, 70%, 80%, 90%, or 100% complementary to the seed regionof the crRNA. The target nucleic acid can be less than about 50%, 60%,70%, 80%, 90%, or 100% complementary to the seed region of the crRNA.

The spacer segment of a nucleic acid-targeting nucleic acid can bemodified (e.g., by genetic engineering) to hybridize to any desiredsequence within a target nucleic acid. For example, a spacer can beengineered (e.g., designed, programmed) to hybridize to a sequence intarget nucleic acid that is involved in cancer, cell growth, DNAreplication, DNA repair, HLA genes, cell surface proteins, T-cellreceptors, immunoglobulin superfamily genes, tumor suppressor genes,microRNA genes, long non-coding RNA genes, transcription factors,globins, viral proteins, mitochondrial genes, and the like.

A spacer sequence can be identified using a computer program (e.g.,machine readable code). The computer program can use variables such aspredicted melting temperature, secondary structure formation, andpredicted annealing temperature, sequence identity, genomic context,chromatin accessibility, % GC, frequency of genomic occurrence,methylation status, presence of SNPs, and the like.

Minimum CRISPR Repeat Sequence

A minimum CRISPR repeat sequence can be a sequence at least about 30%,40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequenceidentity and/or sequence homology with a reference CRISPR repeatsequence (e.g., crRNA from S. pyogenes). A minimum CRISPR repeatsequence can be a sequence with at most about 30%, 40%, 50%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity and/or sequencehomology with a reference CRISPR repeat sequence (e.g., crRNA from S.pyogenes). A minimum CRISPR repeat can comprise nucleotides that canhybridize to a minimum tracrRNA sequence. A minimum CRISPR repeat and aminimum tracrRNA sequence can form a base-paired, double-strandedstructure. Together, the minimum CRISPR repeat and the minimum tracrRNAsequence can facilitate binding to the site-directed polypeptide. A partof the minimum CRISPR repeat sequence can hybridize to the minimumtracrRNA sequence. A part of the minimum CRISPR repeat sequence can beat least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% complementary to the minimum tracrRNA sequence. A part of theminimum CRISPR repeat sequence can be at most about 30%, 40%, 50%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the minimumtracrRNA sequence.

The minimum CRISPR repeat sequence can have a length of from about 6nucleotides to about 100 nucleotides. For example, the minimum CRISPRrepeat sequence can have a length of from about 6 nucleotides (nt) toabout 50 nt, from about 6 nt to about 40 nt, from about 6 nt to about 30nt, from about 6 nt to about 25 nt, from about 6 nt to about 20 nt, fromabout 6 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8nt to about 30 nt, from about 8 nt to about 25 nt, from about 8 nt toabout 20 nt or from about 8 nt to about 15 nt, from about 15 nt to about100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt orfrom about 15 nt to about 25 nt. In some embodiments, the minimum CRISPRrepeat sequence has a length of approximately 12 nucleotides.

The minimum CRISPR repeat sequence can be at least about 60% identicalto a reference minimum CRISPR repeat sequence (e.g., wild type crRNAfrom S. pyogenes) over a stretch of at least 6, 7, or 8 contiguousnucleotides. The minimum CRISPR repeat sequence can be at least about60% identical to a reference minimum CRISPR repeat sequence (e.g., wildtype crRNA from S. pyogenes) over a stretch of at least 6, 7, or 8contiguous nucleotides. For example, the minimum CRISPR repeat sequencecan be at least about 65% identical, at least about 70% identical, atleast about 75% identical, at least about 80% identical, at least about85% identical, at least about 90% identical, at least about 95%identical, at least about 98% identical, at least about 99% identical or100% identical to a reference minimum CRISPR repeat sequence over astretch of at least 6, 7, or 8 contiguous nucleotides.

Minimum tracrRNA Sequence

A minimum tracrRNA sequence can be a sequence with at least about 30%,40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequenceidentity and/or sequence homology to a reference tracrRNA sequence(e.g., wild type tracrRNA from S. pyogenes). A minimum tracrRNA sequencecan be a sequence with at most about 30%, 40%, 50%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% sequence identity and/or sequence homologyto a reference tracrRNA sequence (e.g., wild type tracrRNA from S.pyogenes). A minimum tracrRNA sequence can comprise nucleotides that canhybridize to a minimum CRISPR repeat sequence. A minimum tracrRNAsequence and a minimum CRISPR repeat sequence can form a base-paired,double-stranded structure. Together, the minimum tracrRNA sequence andthe minimum CRISPR repeat can facilitate binding to the site-directedpolypeptide. A part of the minimum tracrRNA sequence can hybridize tothe minimum CRISPR repeat sequence. A part of the minimum tracrRNAsequence can be 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,or 100% complementary to the minimum CRISPR repeat sequence.

The minimum tracrRNA sequence can have a length of from about 6nucleotides to about 100 nucleotides. For example, the minimum tracrRNAsequence can have a length of from about 6 nucleotides (nt) to about 50nt, from about 6 nt to about 40 nt, from about 6 nt to about 30 nt, fromabout 6 nt to about 25 nt, from about 6 nt to about 20 nt, from about 6nt to about 15 nt, from about 8 nt to about 40 nt, from about 8 nt toabout 30 nt, from about 8 nt to about 25 nt, from about 8 nt to about 20nt or from about 8 nt to about 15 nt, from about 15 nt to about 100 nt,from about 15 nt to about 80 nt, from about 15 nt to about 50 nt, fromabout 15 nt to about 40 nt, from about 15 nt to about 30 nt or fromabout 15 nt to about 25 nt. In some embodiments, the minimum tracrRNAsequence has a length of approximately 14 nucleotides.

The minimum tracrRNA sequence can be at least about 60% identical to areference minimum tracrRNA (e.g., wild type, tracrRNA from S. pyogenes)sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.The minimum tracrRNA sequence can be at least about 60% identical to areference minimum tracrRNA (e.g., wild type, tracrRNA from S. pyogenes)sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.For example, the minimum tracrRNA sequence can be at least about 65%identical, at least about 70% identical, at least about 75% identical,at least about 80% identical, at least about 85% identical, at leastabout 90% identical, at least about 95% identical, at least about 98%identical, at least about 99% identical or 100% identical to a referenceminimum tracrRNA sequence over a stretch of at least 6, 7, or 8contiguous nucleotides.

The duplex (i.e., first duplex in FIG. 1B) between the minimum CRISPRRNA and the minimum tracrRNA can comprise a double helix. The first baseof the first strand of the duplex (e.g., the minimum CRISPR repeat inFIG. 1B) can be a guanine. The first base of the first strand of theduplex (e.g., the minimum CRISPR repeat in FIG. 1B) can be an adenine.The duplex (i.e., first duplex in FIG. 1B) between the minimum CRISPRRNA and the minimum tracrRNA can comprise at least about 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more nucleotides. The duplex (i.e., first duplex inFIG. 1B) between the minimum CRISPR RNA and the minimum tracrRNA cancomprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or morenucleotides.

The duplex can comprise a mismatch. The duplex can comprise at leastabout 1, 2, 3, 4, or 5 or mismatches. The duplex can comprise at mostabout 1, 2, 3, 4, or 5 or mismatches. In some instances, the duplexcomprises no more than 2 mismatches.

Bulge

A bulge can refer to an unpaired region of nucleotides within the duplexmade up of the minimum CRISPR repeat and the minimum tracrRNA sequence.The bulge can be important in the binding to the site-directedpolypeptide. A bulge can comprise, on one side of the duplex, anunpaired 5′-XXXY-3′ where X is any purine and Y can be a nucleotide thatcan form a wobble pair with a nucleotide on the opposite strand, and anunpaired nucleotide region on the other side of the duplex.

For example, the bulge can comprise an unpaired purine (e.g., adenine)on the minimum CRISPR repeat strand of the bulge. In some embodiments, abulge can comprise an unpaired 5′-AAGY-3′ of the minimum tracrRNAsequence strand of the bulge, where Y can be a nucleotide that can forma wobble pairing with a nucleotide on the minimum CRISPR repeat strand.

A bulge on a first side of the duplex (e.g., the minimum CRISPR repeatside) can comprise at least 1, 2, 3, 4, or 5 or more unpairednucleotides. A bulge on a first side of the duplex (e.g., the minimumCRISPR repeat side) can comprise at most 1, 2, 3, 4, or 5 or moreunpaired nucleotides. A bulge on the first side of the duplex (e.g., theminimum CRISPR repeat side) can comprise 1 unpaired nucleotide.

A bulge on a second side of the duplex (e.g., the minimum tracrRNAsequence side of the duplex) can comprise at least 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or more unpaired nucleotides. A bulge on a second side ofthe duplex (e.g., the minimum tracrRNA sequence side of the duplex) cancomprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpairednucleotides. A bulge on a second side of the duplex (e.g., the minimumtracrRNA sequence side of the duplex) can comprise 4 unpairednucleotides.

Regions of different numbers of unpaired nucleotides on each strand ofthe duplex can be paired together. For example, a bulge can comprise 5unpaired nucleotides from a first strand and 1 unpaired nucleotide froma second strand. A bulge can comprise 4 unpaired nucleotides from afirst strand and 1 unpaired nucleotide from a second strand. A bulge cancomprise 3 unpaired nucleotides from a first strand and 1 unpairednucleotide from a second strand. A bulge can comprise 2 unpairednucleotides from a first strand and 1 unpaired nucleotide from a secondstrand. A bulge can comprise 1 unpaired nucleotide from a first strandand 1 unpaired nucleotide from a second strand. A bulge can comprise 1unpaired nucleotide from a first strand and 2 unpaired nucleotides froma second strand. A bulge can comprise 1 unpaired nucleotide from a firststrand and 3 unpaired nucleotides from a second strand. A bulge cancomprise 1 unpaired nucleotide from a first strand and 4 unpairednucleotides from a second strand. A bulge can comprise 1 unpairednucleotide from a first strand and 5 unpaired nucleotides from a secondstrand.

In some instances a bulge can comprise at least one wobble pairing. Insome instances, a bulge can comprise at most one wobble pairing. A bulgesequence can comprise at least one purine nucleotide. A bulge sequencecan comprise at least 3 purine nucleotides. A bulge sequence cancomprise at least 5 purine nucleotides. A bulge sequence can comprise atleast one guanine nucleotide. A bulge sequence can comprise at least oneadenine nucleotide.

P-Domain (P-DOMAIN)

A P-domain can refer to a region of a nucleic acid-targeting nucleicacid that can recognize a protospacer adjacent motif (PAM) in a targetnucleic acid. A P-domain can hybridize to a PAM in a target nucleicacid. As such, a P-domain can comprise a sequence that is complementaryto a PAM. A P-domain can be located 3′ to the minimum tracrRNA sequence.A P-domain can be located within a 3′ tracrRNA sequence (i.e., amid-tracrRNA sequence).

A p start at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 ormore nucleotides 3′ of the last paired nucleotide in the minimum CRISPRrepeat and minimum tracrRNA sequence duplex. A P-domain can start atmost about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides 3′ of thelast paired nucleotide in the minimum CRISPR repeat and minimum tracrRNAsequence duplex.

A P-domain can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, or 20 or more consecutive nucleotides. A P-domain can comprise atmost about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more consecutivenucleotides.

In some instances, a P-domain can comprise a CC dinucleotide (i.e., twoconsecutive cytosine nucleotides). The CC dinucleotide can interact withthe GG dinucleotide of a PAM, wherein the PAM comprises a 5′-XGG-3′sequence.

A P-domain can be a nucleotide sequence located in the 3′ tracrRNAsequence (i.e., mid-tracrRNA sequence). A P-domain can comprise duplexednucleotides (e.g., nucleotides in a hairpin, hybridized together. Forexample, a P-domain can comprise a CC dinucleotide that is hybridized toa GG dinucleotide in a hairpin duplex of the 3′ tracrRNA sequence (i.e.,mid-tracrRNA sequence). The activity of the P-domain (e.g., the nucleicacid-targeting nucleic acid's ability to target a target nucleic acid)may be regulated by the hybridization state of the P-DOMAIN. Forexample, if the P-domainis hybridized, the nucleic acid-targetingnucleic acid may not recognize its target. If the P-domainisunhybridized the nucleic acid-targeting nucleic acid may recognize itstarget.

The P-domain can interact with P-domain interacting regions within thesite-directed polypeptide. The P-domain can interact with anarginine-rich basic patch in the site-directed polypeptide. The P-domaininteracting regions can interact with a PAM sequence. The P-domain cancomprise a stem loop. The P-domain can comprise a bulge.

3′tracrRNA Sequence

A 3′tracr RNA sequence can be a sequence with at least about 30%, 40%,50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identityand/or sequence homology with a reference tracrRNA sequence (e.g., atracrRNA from S. pyogenes). A 3′tracr RNA sequence can be a sequencewith at most about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% sequence identity and/or sequence homology with a referencetracrRNA sequence (e.g., tracrRNA from S. pyogenes).

The 3′ tracrRNA sequence can have a length of from about 6 nucleotidesto about 100 nucleotides. For example, the 3′ tracrRNA sequence can havea length of from about 6 nucleotides (nt) to about 50 nt, from about 6nt to about 40 nt, from about 6 nt to about 30 nt, from about 6 nt toabout 25 nt, from about 6 nt to about 20 nt, from about 6 nt to about 15nt, from about 8 nt to about 40 nt, from about 8 nt to about 30 nt, fromabout 8 nt to about 25 nt, from about 8 nt to about 20 nt or from about8 nt to about 15 nt, from about 15 nt to about 100 nt, from about 15 ntto about 80 nt, from about 15 nt to about 50 nt, from about 15 nt toabout 40 nt, from about 15 nt to about 30 nt or from about 15 nt toabout 25 nt. In some embodiments, the 3′ tracrRNA sequence has a lengthof approximately 14 nucleotides.

The 3′ tracrRNA sequence can be at least about 60% identical to areference 3′ tracrRNA sequence (e.g., wild type 3′ tracrRNA sequencefrom S. pyogenes) over a stretch of at least 6, 7, or 8 contiguousnucleotides. For example, the 3′ tracrRNA sequence can be at least about60% identical, at least about 65% identical, at least about 70%identical, at least about 75% identical, at least about 80% identical,at least about 85% identical, at least about 90% identical, at leastabout 95% identical, at least about 98% identical, at least about 99%identical, or 100% identical, to a reference 3′ tracrRNA sequence (e.g.,wild type 3′ tracrRNA sequence from S. pyogenes) over a stretch of atleast 6, 7, or 8 contiguous nucleotides.

A 3′ tracrRNA sequence can comprise more than one duplexed region (e.g.,hairpin, hybridized region). A 3′ tracrRNA sequence can comprise twoduplexed regions.

The 3′ tracrRNA sequence can also be referred to as the mid-tracrRNA(See FIG. 1B). The mid-tracrRNA sequence can comprise a stem loopstructure. In other words, the mid-tracrRNA sequence can comprise ahairpin that is different than a second or third stems, as depicted inFIG. 1B. A stem loop structure in the mid-tracrRNA (i.e., 3′ tracrRNA)can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 or morenucleotides. A stem loop structure in the mid-tracrRNA (i.e., 3′tracrRNA) can comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or morenucleotides. The stem loop structure can comprise a functional moiety.For example, the stem loop structure can comprise an aptamer, aribozyme, a protein-interacting hairpin, a CRISPR array, an intron, andan exon. The stem loop structure can comprise at least about 1, 2, 3, 4,or 5 or more functional moieties. The stem loop structure can compriseat most about 1, 2, 3, 4, or 5 or more functional moieties.

The hairpin in the mid-tracrRNA sequence can comprise a P-domain. TheP-domain can comprise a double stranded region in the hairpin.

tracrRNA Extension Sequence

A tracrRNA extension sequence can provide stability and/or provide alocation for modifications of a nucleic acid-targeting nucleic acid. AtracrRNA extension sequence can have a length of from about 1 nucleotideto about 400 nucleotides. A tracrRNA extension sequence can have alength of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340,360, 380, 400 or more nucleotides. A tracrRNA extension sequence canhave a length from about 20 to about 5000 or more nucleotides. AtracrRNA extension sequence can have a length of more than 1000nucleotides. A tracrRNA extension sequence can have a length of lessthan 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120,140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400nucleotides. A tracrRNA extension sequence can have a length of lessthan 1000 nucleotides. A tracrRNA extension sequence can be less than 10nucleotides in length. A tracrRNA extension sequence can be between 10and 30 nucleotides in length. A tracrRNA extension sequence can bebetween 30-70 nucleotides in length.

The tracrRNA extension sequence can comprise a moiety (e.g., stabilitycontrol sequence, ribozyme, endoribonuclease binding sequence). A moietycan influence the stability of a nucleic acid targeting RNA. A moietycan be a transcriptional terminator segment (i.e., a transcriptiontermination sequence). A moiety of a nucleic acid-targeting nucleic acidcan have a total length of from about 10 nucleotides to about 100nucleotides, from about 10 nucleotides (nt) to about 20 nt, from about20 nt to about 30 nt, from about 30 nt to about 40 nt, from about 40 ntto about 50 nt, from about 50 nt to about 60 nt, from about 60 nt toabout 70 nt, from about 70 nt to about 80 nt, from about 80 nt to about90 nt, or from about 90 nt to about 100 nt, from about 15 nucleotides(nt) to about 80 nt, from about 15 nt to about 50 nt, from about 15 ntto about 40 nt, from about 15 nt to about 30 nt or from about 15 nt toabout 25 nt. The moiety can be one that can function in a eukaryoticcell. In some cases, the moiety can be one that can function in aprokaryotic cell. The moiety can be one that can function in both aeukaryotic cell and a prokaryotic cell.

Non-limiting examples of suitable tracrRNA extension moieties include: a3′ polyadenylated tail, a riboswitch sequence (e.g., to allow forregulated stability and/or regulated accessibility by proteins andprotein complexes), a sequence that forms a dsRNA duplex (i.e., ahairpin), a sequence that targets the RNA to a subcellular location(e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), a modification or sequence that provides abinding site for proteins (e.g., proteins that act on DNA, includingtranscriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like) a modification or sequence thatprovides for increased, decreased, and/or controllable stability, or anycombination thereof. A tracrRNA extension sequence can comprise a primerbinding site, a molecular index (e.g., barcode sequence). In someembodiments of the disclosure, the tracrRNA extension sequence cancomprise one or more affinity tags.

Single Guide Nucleic Acid

The nucleic acid-targeting nucleic acid can be a single guide nucleicacid. The single guide nucleic acid can be RNA. A single guide nucleicacid can comprise a linker (i.e. item 120 from FIG. 1A) between theminimum CRISPR repeat sequence and the minimum tracrRNA sequence thatcan be called a single guide connector sequence.

The single guide connector of a single guide nucleic acid can have alength of from about 3 nucleotides to about 100 nucleotides. Forexample, the linker can have a length of from about 3 nucleotides (nt)to about 90 nt, from about 3 nt to about 80 nt, from about 3 nt to about70 nt, from about 3 nt to about 60 nt, from about 3 nt to about 50 nt,from about 3 nt to about 40 nt, from about 3 nt to about 30 nt, fromabout 3 nt to about 20 nt or from about 3 nt to about 10 nt. Forexample, the linker can have a length of from about 3 nt to about 5 nt,from about 5 nt to about 10 nt, from about 10 nt to about 15 nt, fromabout 15 nt to about 20 nt, from about 20 nt to about 25 nt, from about25 nt to about 30 nt, from about 30 nt to about 35 nt, from about 35 ntto about 40 nt, from about 40 nt to about 50 nt, from about 50 nt toabout 60 nt, from about 60 nt to about 70 nt, from about 70 nt to about80 nt, from about 80 nt to about 90 nt, or from about 90 nt to about 100nt. In some embodiments, the linker of a single guide nucleic acid isbetween 4 and 40 nucleotides. A linker can have a length at least about100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, or 7000 or more nucleotides. A linker can have a length atmost about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,5000, 5500, 6000, 6500, or 7000 or more nucleotides.

The linker sequence can comprise a functional moiety. For example, thelinker sequence can comprise an aptamer, a ribozyme, aprotein-interacting hairpin, a CRISPR array, an intron, and an exon. Thelinker sequence can comprise at least about 1, 2, 3, 4, or 5 or morefunctional moieties. The linker sequence can comprise at most about 1,2, 3, 4, or 5 or more functional moieties.

In some embodiments, the single guide connector can connect the 3′ endof the minimum CRISPR repeat to the 5′ end of the minimum tracrRNAsequence. Alternatively, the single guide connector can connect the 3′end of the tracrRNA sequence to the 5′ end of the minimum CRISPR repeat.That is to say, a single guide nucleic acid can comprise a 5′DNA-binding segment linked to a 3′ protein-binding segment. A singleguide nucleic acid can comprise a 5′ protein-binding segment linked to a3′ DNA-binding segment.

A nucleic acid-targeting nucleic acid can comprise a spacer extensionsequence from 10-5000 nucleotides in length; a spacer sequence of 12-30nucleotides in length, wherein the spacer is at least 50% complementaryto a target nucleic acid; a minimum CRISPR repeat comprising at least60% identity to a crRNA from a prokaryote (e.g., S. pyogenes) or phageover 6, 7, or 8 contiguous nucleotides and wherein the minimum CRISPRrepeat has a length from 5-30 nucleotides; a minimum tracrRNA sequencecomprising at least 60% identity to a tracrRNA from a bacterium (e.g.,S. pyogenes) over 6, 7, or 8 contiguous nucleotides and wherein theminimum tracrRNA sequence has a length from 5-30 nucleotides; a linkersequence that links the minimum CRISPR repeat and the minimum tracrRNAand comprises a length from 3-5000 nucleotides; a 3′ tracrRNA thatcomprises at least 60% identity to a tracrRNA from a prokaryote (e.g.,S. pyogenes) or phage over 6, 7, or 8 contiguous nucleotides and whereinthe 3′ tracrRNA comprises a length from 10-20 nucleotides, and comprisesa duplexed region; and/or a tracrRNA extension comprising 10-5000nucleotides in length, or any combination thereof. This nucleicacid-targeting nucleic acid can be referred to as a single guide nucleicacid-targeting nucleic acid.

A nucleic acid-targeting nucleic acid can comprise a spacer extensionsequence from 10-5000 nucleotides in length; a spacer sequence of 12-30nucleotides in length, wherein the spacer is at least 50% complementaryto a target nucleic acid; a duplex comprising 1) a minimum CRISPR repeatcomprising at least 60% identity to a crRNA from a prokaryote (e.g., S.pyogenes) or phage over 6 contiguous nucleotides and wherein the minimumCRISPR repeat has a length from 5-30 nucleotides, 2) a minimum tracrRNAsequence comprising at least 60% identity to a tracrRNA from a bacterium(e.g., S. pyogenes) over 6 contiguous nucleotides and wherein theminimum tracrRNA sequence has a length from 5-30 nucleotides, and 3) abulge wherein the bulge comprises at least 3 unpaired nucleotides on theminimum CRISPR repeat strand of the duplex and at least 1 unpairednucleotide on the minimum tracrRNA sequence strand of the duplex; alinker sequence that links the minimum CRISPR repeat and the minimumtracrRNA and comprises a length from 3-5000 nucleotides; a 3′ tracrRNAthat comprises at least 60% identity to a tracrRNA from a prokaryote(e.g., S. pyogenes) or phage over 6 contiguous nucleotides, wherein the3′ tracrRNA comprises a length from 10-20 nucleotides and comprises aduplexed region; a P-domain that starts from 1-5 nucleotides downstreamof the duplex comprising the minimum CRISPR repeat and the minimumtracrRNA, comprises 1-10 nucleotides, comprises a sequence that canhybridize to a protospacer adjacent motif in a target nucleic acid, canform a hairpin, and is located in the 3′ tracrRNA region; and/or atracrRNA extension comprising 10-5000 nucleotides in length, or anycombination thereof.

Double Guide Nucleic Acid

A nucleic acid-targeting nucleic acid can be a double guide nucleicacid. The double guide nucleic acid can be RNA. The double guide nucleicacid can comprise two separate nucleic acid molecules (i.e.polynucleotides). Each of the two nucleic acid molecules of a doubleguide nucleic acid-targeting nucleic acid can comprise a stretch ofnucleotides that can hybridize to one another such that thecomplementary nucleotides of the two nucleic acid molecules hybridize toform the double stranded duplex of the protein-binding segment. If nototherwise specified, the term “nucleic acid-targeting nucleic acid” canbe inclusive, referring to both single-molecule nucleic acid-targetingnucleic acids and double-molecule nucleic acid-targeting nucleic acids.

A double-guide nucleic acid-targeting nucleic acid can comprise 1) afirst nucleic acid molecule comprising a spacer extension sequence from10-5000 nucleotides in length; a spacer sequence of 12-30 nucleotides inlength, wherein the spacer is at least 50% complementary to a targetnucleic acid; and a minimum CRISPR repeat comprising at least 60%identity to a crRNA from a prokaryote (e.g., S. pyogenes) or phage over6 contiguous nucleotides and wherein the minimum CRISPR repeat has alength from 5-30 nucleotides; and 2) a second nucleic acid molecule ofthe double-guide nucleic acid-targeting nucleic acid can comprise aminimum tracrRNA sequence comprising at least 60% identity to a tracrRNAfrom a prokaryote (e.g., S. pyogenes) or phage over 6 contiguousnucleotides and wherein the minimum tracrRNA sequence has a length from5-30 nucleotides; a 3′ tracrRNA that comprises at least 60% identity toa tracrRNA from a bacterium (e.g., S. pyogenes) over 6 contiguousnucleotides and wherein the 3′ tracrRNA comprises a length from 10-20nucleotides, and comprises a duplexed region; and/or a tracrRNAextension comprising 10-5000 nucleotides in length, or any combinationthereof.

In some instances, a double-guide nucleic acid-targeting nucleic acidcan comprise 1) a first nucleic acid molecule comprising a spacerextension sequence from 10-5000 nucleotides in length; a spacer sequenceof 12-30 nucleotides in length, wherein the spacer is at least 50%complementary to a target nucleic acid; a minimum CRISPR repeatcomprising at least 60% identity to a crRNA from a prokaryote (e.g., S.pyogenes) or phage over 6 contiguous nucleotides and wherein the minimumCRISPR repeat has a length from 5-30 nucleotides, and at least 3unpaired nucleotides of a bulge; and 2) a second nucleic acid moleculeof the double-guide nucleic acid-targeting nucleic acid can comprise aminimum tracrRNA sequence comprising at least 60% identity to a tracrRNAfrom a prokaryote (e.g., S. pyogenes) or phage over 6 contiguousnucleotides and wherein the minimum tracrRNA sequence has a length from5-30 nucleotides and at least 1 unpaired nucleotide of a bulge, whereinthe 1 unpaired nucleotide of the bulge is located in the same bulge asthe 3 unpaired nucleotides of the minimum CRISPR repeat; a 3′ tracrRNAthat comprises at least 60% identity to a tracrRNA from a prokaryote(e.g., S. pyogenes) or phage over 6 contiguous nucleotides and whereinthe 3′ tracrRNA comprises a length from 10-20 nucleotides, and comprisesa duplexed region; a P-domain that starts from 1-5 nucleotidesdownstream of the duplex comprising the minimum CRISPR repeat and theminimum tracrRNA, comprises 1-10 nucleotides, comprises a sequence thatcan hybridize to a protospacer adjacent motif in a target nucleic acid,can form a hairpin, and is located in the 3′ tracrRNA region; and/or atracrRNA extension comprising 10-5000 nucleotides in length, or anycombination thereof.

Complex of a Nucleic Acid-Targeting Nucleic Acid and a Site-DirectedPolypeptide

A nucleic acid-targeting nucleic acid can interact with a site-directedpolypeptide (e.g., a nucleic acid-guided nucleases, Cas9), therebyforming a complex. The nucleic acid-targeting nucleic acid can guide thesite-directed polypeptide to a target nucleic acid.

In some embodiments, a nucleic acid-targeting nucleic acid can beengineered such that the complex (e.g., comprising a site-directedpolypeptide and a nucleic acid-targeting nucleic acid) can bind outsideof the cleavage site of the site-directed polpeptide. In this case, thetarget nucleic acid may not interact with the complex and the targetnucleic acid can be excised (e.g., free from the complex).

In some embodiments, a nucleic acid-targeting nucleic acid can beengineered such that the complex can bind inside of the cleavage site ofthe site-directed polpeptide. In this case, the target nucleic acid caninteract with the complex and the target nucleic acid can be bound(e.g., bound to the complex).

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide can comprise an amino acidsequence comprising at least 15% amino acid identity to a Cas9 from S.pyogenes, and two nucleic acid cleaving domains (i.e., an HNH domain anda RuvC domain); and a double-guide nucleic acid-targeting nucleic acidcomprising 1) a first nucleic acid molecule comprising a spacerextension sequence from 10-5000 nucleotides in length; a spacer sequenceof 12-30 nucleotides in length, wherein the spacer is at least 50%complementary to a target nucleic acid; and a minimum CRISPR repeatcomprising at least 60% identity to a crRNA from a prokaryote (e.g., S.pyogenes) or phage over 6 contiguous nucleotides and wherein the minimumCRISPR repeat has a length from 5-30 nucleotides; and 2) a secondnucleic acid molecule of the double-guide nucleic acid-targeting nucleicacid can comprise a minimum tracrRNA sequence comprising at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides; a 3′ tracrRNA that comprises atleast 60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)over 6 contiguous nucleotides and wherein the 3′ tracrRNA comprises alength from 10-20 nucleotides, and comprises a duplexed region; and/or atracrRNA extension comprising 10-5000 nucleotides in length, or anycombination thereof.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide can comprise an amino acidsequence comprising at least 15% amino acid identity to a Cas9 from S.pyogenes, and two nucleic acid cleaving domains (i.e., an HNH domain anda RuvC domain); and, a double-guide nucleic acid-targeting nucleic acidcomprising 1) a first nucleic acid molecule comprising a spacerextension sequence from 10-5000 nucleotides in length; a spacer sequenceof 12-30 nucleotides in length, wherein the spacer is at least 50%complementary to a target nucleic acid; a minimum CRISPR repeatcomprising at least 60% identity to a crRNA from a prokaryote (e.g., S.pyogenes) or phage over 6 contiguous nucleotides and wherein the minimumCRISPR repeat has a length from 5-30 nucleotides, and at least 3unpaired nucleotides of a bulge; and 2) a second nucleic acid moleculeof the double-guide nucleic acid-targeting nucleic acid can comprise aminimum tracrRNA sequence comprising at least 60% identity to a tracrRNAfrom a prokaryote (e.g., S. pyogenes) or phage over 6 contiguousnucleotides and wherein the minimum tracrRNA sequence has a length from5-30 nucleotides and at least 1 unpaired nucleotide of a bulge, whereinthe 1 unpaired nucleotide of the bulge is located in the same bulge asthe 3 unpaired nucleotides of the minimum CRISPR repeat; a 3′ tracrRNAthat comprises at least 60% identity to a tracrRNA from a prokaryote(e.g., S. pyogenes) or phage over 6 contiguous nucleotides and whereinthe 3′ tracrRNA comprises a length from 10-20 nucleotides, and comprisesa duplexed region; a P-domain that starts from 1-5 nucleotidesdownstream of the duplex comprising the minimum CRISPR repeat and theminimum tracrRNA, comprises 1-10 nucleotides, comprises a sequence thatcan hybridize to a protospacer adjacent motif in a target nucleic acid,can form a hairpin, and is located in the 3′ tracrRNA region; and/or atracrRNA extension comprising 10-5000 nucleotides in length, or anycombination thereof.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide can comprise an amino acidsequence comprising at least 15% amino acid identity to a Cas9 from S.pyogenes and, two nucleic acid cleaving domains (i.e., an HNH domain anda RuvC domain); and, a nucleic acid-targeting nucleic acid comprising aspacer extension sequence from 10-5000 nucleotides in length; a spacersequence of 12-30 nucleotides in length, wherein the spacer is at least50% complementary to a target nucleic acid; a minimum CRISPR repeatcomprising at least 60% identity to a crRNA from a prokaryote (e.g., S.pyogenes) or phage over 6, 7, or 8 contiguous nucleotides and whereinthe minimum CRISPR repeat has a length from 5-30 nucleotides; a minimumtracrRNA sequence comprising at least 60% identity to a tracrRNA from abacterium (e.g., S. pyogenes) over 6, 7, or 8 contiguous nucleotides andwherein the minimum tracrRNA sequence has a length from 5-30nucleotides; a linker sequence that links the minimum CRISPR repeat andthe minimum tracrRNA and comprises a length from 3-5000 nucleotides; a3′ tracrRNA that comprises at least 60% identity to a tracrRNA from aprokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguousnucleotides and wherein the 3′ tracrRNA comprises a length from 10-20nucleotides, and comprises a duplexed region; and/or a tracrRNAextension comprising 10-5000 nucleotides in length, or any combinationthereof. This nucleic acid-targeting nucleic acid can be referred to asa single guide nucleic acid-targeting nucleic acid.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide can comprise an amino acidsequence comprising at least 15% amino acid identity to a Cas9 from S.pyogenes, and two nucleic acid cleaving domains (i.e., an HNH domain anda RuvC domain); and, a nucleic acid-targeting nucleic acid cancomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a duplexcomprising 1) a minimum CRISPR repeat comprising at least 60% identityto a crRNA from a prokaryote (e.g., S. pyogenes) or phage over 6contiguous nucleotides and wherein the minimum CRISPR repeat has alength from 5-30 nucleotides, 2) a minimum tracrRNA sequence comprisingat least 60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)over 6 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides, and 3) a bulge wherein the bulgecomprises at least 3 unpaired nucleotides on the minimum CRISPR repeatstrand of the duplex and at least 1 unpaired nucleotide on the minimumtracrRNA sequence strand of the duplex; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6 contiguous nucleotides, wherein the 3′ tracrRNA comprises alength from 10-20 nucleotides and comprises a duplexed region; aP-domain that starts from 1-5 nucleotides downstream of the duplexcomprising the minimum CRISPR repeat and the minimum tracrRNA, comprises1-10 nucleotides, comprises a sequence that can hybridize to aprotospacer adjacent motif in a target nucleic acid, can form a hairpin,and is located in the 3′ tracrRNA region; and/or a tracrRNA extensioncomprising 10-5000 nucleotides in length, or any combination thereof. Insome instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide comprises an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from S. pyogenes,two nucleic acid cleaving domains (i.e., an HNH domain and a RuvCdomain), and a linker linking the site-directed polypeptide to anon-native sequence; and a double-guide nucleic acid-targeting nucleicacid comprising 1) a first nucleic acid molecule comprising a spacerextension sequence from 10-5000 nucleotides in length; a spacer sequenceof 12-30 nucleotides in length, wherein the spacer is at least 50%complementary to a target nucleic acid; and a minimum CRISPR repeatcomprising at least 60% identity to a crRNA from a prokaryote (e.g., S.pyogenes) or phage over 6 contiguous nucleotides and wherein the minimumCRISPR repeat has a length from 5-30 nucleotides; and 2) a secondnucleic acid molecule of the double-guide nucleic acid-targeting nucleicacid can comprise a minimum tracrRNA sequence comprising at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides; a 3′ tracrRNA that comprises atleast 60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)over 6 contiguous nucleotides and wherein the 3′ tracrRNA comprises alength from 10-20 nucleotides, and comprises a duplexed region; and/or atracrRNA extension comprising 10-5000 nucleotides in length, or anycombination thereof.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide comprises an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from S. pyogenes,two nucleic acid cleaving domains (i.e., an HNH domain and a RuvCdomain), and a linker linking the site-directed polypeptide to anon-native sequence; and a double-guide nucleic acid-targeting nucleicacid comprising 1) a first nucleic acid molecule comprising a spacerextension sequence from 10-5000 nucleotides in length; a spacer sequenceof 12-30 nucleotides in length, wherein the spacer is at least 50%complementary to a target nucleic acid; a minimum CRISPR repeatcomprising at least 60% identity to a crRNA from a prokaryote (e.g., S.pyogenes) or phage over 6 contiguous nucleotides and wherein the minimumCRISPR repeat has a length from 5-30 nucleotides, and at least 3unpaired nucleotides of a bulge; and 2) a second nucleic acid moleculeof the double-guide nucleic acid-targeting nucleic acid can comprise aminimum tracrRNA sequence comprising at least 60% identity to a tracrRNAfrom a prokaryote (e.g., S. pyogenes) or phage over 6 contiguousnucleotides and wherein the minimum tracrRNA sequence has a length from5-30 nucleotides and at least 1 unpaired nucleotide of a bulge, whereinthe 1 unpaired nucleotide of the bulge is located in the same bulge asthe 3 unpaired nucleotides of the minimum CRISPR repeat; a 3′ tracrRNAthat comprises at least 60% identity to a tracrRNA from a prokaryote(e.g., S. pyogenes) or phage over 6 contiguous nucleotides and whereinthe 3′ tracrRNA comprises a length from 10-20 nucleotides, and comprisesa duplexed region; a P-domain that starts from 1-5 nucleotidesdownstream of the duplex comprising the minimum CRISPR repeat and theminimum tracrRNA, comprises 1-10 nucleotides, comprises a sequence thatcan hybridize to a protospacer adjacent motif in a target nucleic acid,can form a hairpin, and is located in the 3′ tracrRNA region; and/or atracrRNA extension comprising 10-5000 nucleotides in length, or anycombination thereof.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide comprises an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from S. pyogenes,two nucleic acid cleaving domains (i.e., an HNH domain and a RuvCdomain), and a linker linking the site-directed polypeptide to anon-native sequence; and a nucleic acid-targeting nucleic acidcomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a minimumCRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguousnucleotides and wherein the minimum CRISPR repeat has a length from 5-30nucleotides; a minimum tracrRNA sequence comprising at least 60%identity to a tracrRNA from a bacterium (e.g., S. pyogenes) over 6, 7,or 8 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6, 7, or 8 contiguous nucleotides and wherein the 3′ tracrRNAcomprises a length from 10-20 nucleotides, and comprises a duplexedregion; and/or a tracrRNA extension comprising 10-5000 nucleotides inlength, or any combination thereof. This nucleic acid-targeting nucleicacid can be referred to as a single guide nucleic acid-targeting nucleicacid.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide comprises an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from S. pyogenes,two nucleic acid cleaving domains (i.e., an HNH domain and a RuvCdomain), and a linker linking the site-directed polypeptide to anon-native sequence; and nucleic acid-targeting nucleic acid cancomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a duplexcomprising 1) a minimum CRISPR repeat comprising at least 60% identityto a crRNA from a prokaryote (e.g., S. pyogenes) or phage over 6contiguous nucleotides and wherein the minimum CRISPR repeat has alength from 5-30 nucleotides, 2) a minimum tracrRNA sequence comprisingat least 60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)over 6 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides, and 3) a bulge wherein the bulgecomprises at least 3 unpaired nucleotides on the minimum CRISPR repeatstrand of the duplex and at least 1 unpaired nucleotide on the minimumtracrRNA sequence strand of the duplex; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6 contiguous nucleotides, wherein the 3′ tracrRNA comprises alength from 10-20 nucleotides and comprises a duplexed region; aP-domain that starts from 1-5 nucleotides downstream of the duplexcomprising the minimum CRISPR repeat and the minimum tracrRNA, comprises1-10 nucleotides, comprises a sequence that can hybridize to aprotospacer adjacent motif in a target nucleic acid, can form a hairpin,and is located in the 3′ tracrRNA region; and/or a tracrRNA extensioncomprising 10-5000 nucleotides in length, or any combination thereof.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide comprises at least 15% amino acididentity to a Cas9 from S. pyogenes, two nucleic acid cleaving domains(i.e., an HNH domain and a RuvC domain), wherein the site-directedpolypeptide comprises a mutation in one or both of the nucleic acidcleaving domains that reduces the cleaving activity of the nucleasedomains by at least 50%; and a double-guide nucleic acid-targetingnucleic acid comprising 1) a first nucleic acid molecule comprising aspacer extension sequence from 10-5000 nucleotides in length; a spacersequence of 12-30 nucleotides in length, wherein the spacer is at least50% complementary to a target nucleic acid; and a minimum CRISPR repeatcomprising at least 60% identity to a crRNA from a prokaryote (e.g., S.pyogenes) or phage over 6 contiguous nucleotides and wherein the minimumCRISPR repeat has a length from 5-30 nucleotides; and 2) a secondnucleic acid molecule of the double-guide nucleic acid-targeting nucleicacid can comprise a minimum tracrRNA sequence comprising at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides; a 3′ tracrRNA that comprises atleast 60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)over 6 contiguous nucleotides and wherein the 3′ tracrRNA comprises alength from 10-20 nucleotides, and comprises a duplexed region; and/or atracrRNA extension comprising 10-5000 nucleotides in length, or anycombination thereof.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide comprises at least 15% amino acididentity to a Cas9 from S. pyogenes, two nucleic acid cleaving domains(i.e., an HNH domain and a RuvC domain), wherein the site-directedpolypeptide comprises a mutation in one or both of the nucleic acidcleaving domains that reduces the cleaving activity of the nucleasedomains by at least 50%; and a double-guide nucleic acid-targetingnucleic acid comprising 1) a first nucleic acid molecule comprising aspacer extension sequence from 10-5000 nucleotides in length; a spacersequence of 12-30 nucleotides in length, wherein the spacer is at least50% complementary to a target nucleic acid; a minimum CRISPR repeatcomprising at least 60% identity to a crRNA from a prokaryote (e.g., S.pyogenes) or phage over 6 contiguous nucleotides and wherein the minimumCRISPR repeat has a length from 5-30 nucleotides, and at least 3unpaired nucleotides of a bulge; and 2) a second nucleic acid moleculeof the double-guide nucleic acid-targeting nucleic acid can comprise aminimum tracrRNA sequence comprising at least 60% identity to a tracrRNAfrom a prokaryote (e.g., S. pyogenes) or phage over 6 contiguousnucleotides and wherein the minimum tracrRNA sequence has a length from5-30 nucleotides and at least 1 unpaired nucleotide of a bulge, whereinthe 1 unpaired nucleotide of the bulge is located in the same bulge asthe 3 unpaired nucleotides of the minimum CRISPR repeat; a 3′ tracrRNAthat comprises at least 60% identity to a tracrRNA from a prokaryote(e.g., S. pyogenes) or phage over 6 contiguous nucleotides and whereinthe 3′ tracrRNA comprises a length from 10-20 nucleotides, and comprisesa duplexed region; a P-domain that starts from 1-5 nucleotidesdownstream of the duplex comprising the minimum CRISPR repeat and theminimum tracrRNA, comprises 1-10 nucleotides, comprises a sequence thatcan hybridize to a protospacer adjacent motif in a target nucleic acid,can form a hairpin, and is located in the 3′ tracrRNA region; and/or atracrRNA extension comprising 10-5000 nucleotides in length, or anycombination thereof.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide comprises at least 15% amino acididentity to a Cas9 from S. pyogenes, two nucleic acid cleaving domains(i.e., an HNH domain and a RuvC domain), wherein the site-directedpolypeptide comprises a mutation in one or both of the nucleic acidcleaving domains that reduces the cleaving activity of the nucleasedomains by at least 50%; and nucleic acid-targeting nucleic acidcomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a minimumCRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguousnucleotides and wherein the minimum CRISPR repeat has a length from 5-30nucleotides; a minimum tracrRNA sequence comprising at least 60%identity to a tracrRNA from a bacterium (e.g., S. pyogenes) over 6, 7,or 8 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6, 7, or 8 contiguous nucleotides and wherein the 3′ tracrRNAcomprises a length from 10-20 nucleotides, and comprises a duplexedregion; and/or a tracrRNA extension comprising 10-5000 nucleotides inlength, or any combination thereof. This nucleic acid-targeting nucleicacid can be referred to as a single guide nucleic acid-targeting nucleicacid.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide comprises at least 15% amino acididentity to a Cas9 from S. pyogenes, two nucleic acid cleaving domains(i.e., an HNH domain and a RuvC domain), wherein the site-directedpolypeptide comprises a mutation in one or both of the nucleic acidcleaving domains that reduces the cleaving activity of the nucleasedomains by at least 50%; and nucleic acid-targeting nucleic acid cancomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a duplexcomprising 1) a minimum CRISPR repeat comprising at least 60% identityto a crRNA from a prokaryote (e.g., S. pyogenes) or phage over 6contiguous nucleotides and wherein the minimum CRISPR repeat has alength from 5-30 nucleotides, 2) a minimum tracrRNA sequence comprisingat least 60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)over 6 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides, and 3) a bulge wherein the bulgecomprises at least 3 unpaired nucleotides on the minimum CRISPR repeatstrand of the duplex and at least 1 unpaired nucleotide on the minimumtracrRNA sequence strand of the duplex; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6 contiguous nucleotides, wherein the 3′ tracrRNA comprises alength from 10-20 nucleotides and comprises a duplexed region; aP-domain that starts from 1-5 nucleotides downstream of the duplexcomprising the minimum CRISPR repeat and the minimum tracrRNA, comprises1-10 nucleotides, comprises a sequence that can hybridize to aprotospacer adjacent motif in a target nucleic acid, can form a hairpin,and is located in the 3′ tracrRNA region; and/or a tracrRNA extensioncomprising 10-5000 nucleotides in length, or any combination thereof.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide comprises at least 15% amino acididentity to a Cas9 from S. pyogenes, two nucleic acid cleaving domains(i.e., an HNH domain and a RuvC domain), wherein the site-directedpolypeptide comprises a mutation in one or both of the nucleic acidcleaving domains that reduces the cleaving activity of the nucleasedomains by at least 50%; and a double-guide nucleic acid-targetingnucleic acid comprising 1) a first nucleic acid molecule comprising aspacer extension sequence from 10-5000 nucleotides in length; a spacersequence of 12-30 nucleotides in length, wherein the spacer is at least50% complementary to a target nucleic acid; and a minimum CRISPR repeatcomprising at least 60% identity to a crRNA from a prokaryote (e.g., S.pyogenes) or phage over 6 contiguous nucleotides and wherein the minimumCRISPR repeat has a length from 5-30 nucleotides; and 2) a secondnucleic acid molecule of the double-guide nucleic acid-targeting nucleicacid can comprise a minimum tracrRNA sequence comprising at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides; a 3′ tracrRNA that comprises atleast 60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)over 6 contiguous nucleotides and wherein the 3′ tracrRNA comprises alength from 10-20 nucleotides, and comprises a duplexed region; and/or atracrRNA extension comprising 10-5000 nucleotides in length, or anycombination thereof.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide comprises at least 15% amino acididentity to a Cas9 from S. pyogenes, two nucleic acid cleaving domains(i.e., an HNH domain and a RuvC domain), wherein the site-directedpolypeptide comprises a mutation in one or both of the nucleic acidcleaving domains that reduces the cleaving activity of the nucleasedomains by at least 50%; and a double-guide nucleic acid-targetingnucleic acid comprising 1) a first nucleic acid molecule comprising aspacer extension sequence from 10-5000 nucleotides in length; a spacersequence of 12-30 nucleotides in length, wherein the spacer is at least50% complementary to a target nucleic acid; a minimum CRISPR repeatcomprising at least 60% identity to a crRNA from a prokaryote (e.g., S.pyogenes) or phage over 6 contiguous nucleotides and wherein the minimumCRISPR repeat has a length from 5-30 nucleotides, and at least 3unpaired nucleotides of a bulge; and 2) a second nucleic acid moleculeof the double-guide nucleic acid-targeting nucleic acid can comprise aminimum tracrRNA sequence comprising at least 60% identity to a tracrRNAfrom a prokaryote (e.g., S. pyogenes) or phage over 6 contiguousnucleotides and wherein the minimum tracrRNA sequence has a length from5-30 nucleotides and at least 1 unpaired nucleotide of a bulge, whereinthe 1 unpaired nucleotide of the bulge is located in the same bulge asthe 3 unpaired nucleotides of the minimum CRISPR repeat; a 3′ tracrRNAthat comprises at least 60% identity to a tracrRNA from a prokaryote(e.g., S. pyogenes) or phage over 6 contiguous nucleotides and whereinthe 3′ tracrRNA comprises a length from 10-20 nucleotides, and comprisesa duplexed region; a P-domain that starts from 1-5 nucleotidesdownstream of the duplex comprising the minimum CRISPR repeat and theminimum tracrRNA, comprises 1-10 nucleotides, comprises a sequence thatcan hybridize to a protospacer adjacent motif in a target nucleic acid,can form a hairpin, and is located in the 3′ tracrRNA region; and/or atracrRNA extension comprising 10-5000 nucleotides in length, or anycombination thereof.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide comprises at least 15% amino acididentity to a Cas9 from S. pyogenes, two nucleic acid cleaving domains(i.e., an HNH domain and a RuvC domain), wherein the site-directedpolypeptide comprises a mutation in one or both of the nucleic acidcleaving domains that reduces the cleaving activity of the nucleasedomains by at least 50%; and nucleic acid-targeting nucleic acidcomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a minimumCRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguousnucleotides and wherein the minimum CRISPR repeat has a length from 5-30nucleotides; a minimum tracrRNA sequence comprising at least 60%identity to a tracrRNA from a bacterium (e.g., S. pyogenes) over 6, 7,or 8 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6, 7, or 8 contiguous nucleotides and wherein the 3′ tracrRNAcomprises a length from 10-20 nucleotides, and comprises a duplexedregion; and/or a tracrRNA extension comprising 10-5000 nucleotides inlength, or any combination thereof. This nucleic acid-targeting nucleicacid can be referred to as a single guide nucleic acid-targeting nucleicacid.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide comprises at least 15% amino acididentity to a Cas9 from S. pyogenes, two nucleic acid cleaving domains(i.e., an HNH domain and a RuvC domain), wherein the site-directedpolypeptide comprises a mutation in one or both of the nucleic acidcleaving domains that reduces the cleaving activity of the nucleasedomains by at least 50%; and a nucleic acid-targeting nucleic acid cancomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a duplexcomprising 1) a minimum CRISPR repeat comprising at least 60% identityto a crRNA from a prokaryote (e.g., S. pyogenes) or phage over 6contiguous nucleotides and wherein the minimum CRISPR repeat has alength from 5-30 nucleotides, 2) a minimum tracrRNA sequence comprisingat least 60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)over 6 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides, and 3) a bulge wherein the bulgecomprises at least 3 unpaired nucleotides on the minimum CRISPR repeatstrand of the duplex and at least 1 unpaired nucleotide on the minimumtracrRNA sequence strand of the duplex; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6 contiguous nucleotides, wherein the 3′ tracrRNA comprises alength from 10-20 nucleotides and comprises a duplexed region; aP-domain that starts from 1-5 nucleotides downstream of the duplexcomprising the minimum CRISPR repeat and the minimum tracrRNA, comprises1-10 nucleotides, comprises a sequence that can hybridize to aprotospacer adjacent motif in a target nucleic acid, can form a hairpin,and is located in the 3′ tracrRNA region; and/or a tracrRNA extensioncomprising 10-5000 nucleotides in length, or any combination thereof.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide comprises at least 15% amino acididentity to a Cas9 from S. pyogenes, two nucleic acid cleaving domains(i.e., an HNH domain and a RuvC domain), wherein the site-directedpolypeptide comprises a mutation in one or both of the nucleic acidcleaving domains that reduces the cleaving activity of the nucleasedomains by at least 50%; and a double-guide nucleic acid-targetingnucleic acid comprising 1) a first nucleic acid molecule comprising aspacer extension sequence from 10-5000 nucleotides in length; a spacersequence of 12-30 nucleotides in length, wherein the spacer is at least50% complementary to a target nucleic acid; and a minimum CRISPR repeatcomprising at least 60% identity to a crRNA from a prokaryote (e.g., S.pyogenes) or phage over 6 contiguous nucleotides and wherein the minimumCRISPR repeat has a length from 5-30 nucleotides; and 2) a secondnucleic acid molecule of the double-guide nucleic acid-targeting nucleicacid can comprise a minimum tracrRNA sequence comprising at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides; a 3′ tracrRNA that comprises atleast 60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)over 6 contiguous nucleotides and wherein the 3′ tracrRNA comprises alength from 10-20 nucleotides, and comprises a duplexed region; and/or atracrRNA extension comprising 10-5000 nucleotides in length, or anycombination thereof.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide comprises at least 15% amino acididentity to a Cas9 from S. pyogenes, and two nucleic acid cleavingdomains, wherein one or both of the nucleic acid cleaving domainscomprise at least 50% amino acid identity to a nuclease domain from Cas9from S. pyogenes; and a double-guide nucleic acid-targeting nucleic acidcomprising 1) a first nucleic acid molecule comprising a spacerextension sequence from 10-5000 nucleotides in length; a spacer sequenceof 12-30 nucleotides in length, wherein the spacer is at least 50%complementary to a target nucleic acid; a minimum CRISPR repeatcomprising at least 60% identity to a crRNA from a prokaryote (e.g., S.pyogenes) or phage over 6 contiguous nucleotides and wherein the minimumCRISPR repeat has a length from 5-30 nucleotides, and at least 3unpaired nucleotides of a bulge; and 2) a second nucleic acid moleculeof the double-guide nucleic acid-targeting nucleic acid can comprise aminimum tracrRNA sequence comprising at least 60% identity to a tracrRNAfrom a prokaryote (e.g., S. pyogenes) or phage over 6 contiguousnucleotides and wherein the minimum tracrRNA sequence has a length from5-30 nucleotides and at least 1 unpaired nucleotide of a bulge, whereinthe 1 unpaired nucleotide of the bulge is located in the same bulge asthe 3 unpaired nucleotides of the minimum CRISPR repeat; a 3′ tracrRNAthat comprises at least 60% identity to a tracrRNA from a prokaryote(e.g., S. pyogenes) or phage over 6 contiguous nucleotides and whereinthe 3′ tracrRNA comprises a length from 10-20 nucleotides, and comprisesa duplexed region; a P-domain that starts from 1-5 nucleotidesdownstream of the duplex comprising the minimum CRISPR repeat and theminimum tracrRNA, comprises 1-10 nucleotides, comprises a sequence thatcan hybridize to a protospacer adjacent motif in a target nucleic acid,can form a hairpin, and is located in the 3′ tracrRNA region; and/or atracrRNA extension comprising 10-5000 nucleotides in length, or anycombination thereof.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide comprises at least 15% amino acididentity to a Cas9 from S. pyogenes, and two nucleic acid cleavingdomains, wherein one or both of the nucleic acid cleaving domainscomprise at least 50% amino acid identity to a nuclease domain from Cas9from S. pyogenes; and nucleic acid-targeting nucleic acid comprising aspacer extension sequence from 10-5000 nucleotides in length; a spacersequence of 12-30 nucleotides in length, wherein the spacer is at least50% complementary to a target nucleic acid; a minimum CRISPR repeatcomprising at least 60% identity to a crRNA from a prokaryote (e.g., S.pyogenes) or phage over 6, 7, or 8 contiguous nucleotides and whereinthe minimum CRISPR repeat has a length from 5-30 nucleotides; a minimumtracrRNA sequence comprising at least 60% identity to a tracrRNA from abacterium (e.g., S. pyogenes) over 6, 7, or 8 contiguous nucleotides andwherein the minimum tracrRNA sequence has a length from 5-30nucleotides; a linker sequence that links the minimum CRISPR repeat andthe minimum tracrRNA and comprises a length from 3-5000 nucleotides; a3′ tracrRNA that comprises at least 60% identity to a tracrRNA from aprokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguousnucleotides and wherein the 3′ tracrRNA comprises a length from 10-20nucleotides, and comprises a duplexed region; and/or a tracrRNAextension comprising 10-5000 nucleotides in length, or any combinationthereof. This nucleic acid-targeting nucleic acid can be referred to asa single guide nucleic acid-targeting nucleic acid.

In some instances, a complex can comprise a site-directed polypeptide,wherein the site-directed polypeptide comprises at least 15% amino acididentity to a Cas9 from S. pyogenes, and two nucleic acid cleavingdomains, wherein one or both of the nucleic acid cleaving domainscomprise at least 50% amino acid identity to a nuclease domain from Cas9from S. pyogenes; and a nucleic acid-targeting nucleic acid cancomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a duplexcomprising 1) a minimum CRISPR repeat comprising at least 60% identityto a crRNA from a prokaryote (e.g., S. pyogenes) or phage over 6contiguous nucleotides and wherein the minimum CRISPR repeat has alength from 5-30 nucleotides, 2) a minimum tracrRNA sequence comprisingat least 60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)over 6 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides, and 3) a bulge wherein the bulgecomprises at least 3 unpaired nucleotides on the minimum CRISPR repeatstrand of the duplex and at least 1 unpaired nucleotide on the minimumtracrRNA sequence strand of the duplex; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6 contiguous nucleotides, wherein the 3′ tracrRNA comprises alength from 10-20 nucleotides and comprises a duplexed region; aP-domain that starts from 1-5 nucleotides downstream of the duplexcomprising the minimum CRISPR repeat and the minimum tracrRNA, comprises1-10 nucleotides, comprises a sequence that can hybridize to aprotospacer adjacent motif in a target nucleic acid, can form a hairpin,and is located in the 3′ tracrRNA region; and/or a tracrRNA extensioncomprising 10-5000 nucleotides in length, or any combination thereof.

Any nucleic acid-targeting nucleic acid of the disclosure, asite-directed polypeptide of the disclosure, an effector protein, amultiplexed genetic targeting agent, a donor polynucleotide, a tandemfusion protein, a reporter element, a genetic element of interest, acomponent of a split system and/or any nucleic acid or proteinaceousmolecule necessary to carry out the embodiments of the methods of thedisclosure may be recombinant, purified and/or isolated.

Nucleic Acids Encoding a Nucleic Acid-Targeting Nucleic Acid and/or aSite-Directed Polypeptide

The present disclosure provides for a nucleic acid comprising anucleotide sequence encoding a nucleic acid-targeting nucleic acid ofthe disclosure, a site-directed polypeptide of the disclosure, aneffector protein, a multiplexed genetic targeting agent, a donorpolynucleotide, a tandem fusion protein, a reporter element, a geneticelement of interest, a component of a split system and/or any nucleicacid or proteinaceous molecule necessary to carry out the embodiments ofthe methods of the disclosure. In some embodiments, the nucleic acidencoding a nucleic acid-targeting nucleic acid of the disclosure, asite-directed polypeptide of the disclosure, an effector protein, amultiplexed genetic targeting agent, a donor polynucleotide, a tandemfusion protein, a reporter element, a genetic element of interest, acomponent of a split system and/or any nucleic acid or proteinaceousmolecule necessary to carry out the embodiments of the methods of thedisclosure can be a vector (e.g., a recombinant expression vector).

In some embodiments, the recombinant expression vector can be a viralconstruct, (e.g., a recombinant adeno-associated virus construct), arecombinant adenoviral construct, a recombinant lentiviral construct, arecombinant retroviral construct, etc.

Suitable expression vectors can include, but are not limited to, viralvectors (e.g. viral vectors based on vaccinia virus, poliovirus,adenovirus, adeno-associated virus, SV40, herpes simplex virus, humanimmunodeficiency virus, a retroviral vector (e.g., Murine LeukemiaVirus, spleen necrosis virus, and vectors derived from retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, alentivirus, human immunodeficiency virus, myeloproliferative sarcomavirus, and mammary tumor virus), plant vectors (e.g., T-DNA vector), andthe like. The following vectors can be provided by way of example, foreukaryotic host cells: pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40(Pharmacia). Other vectors may be used so long as they are compatiblewith the host cell.

In some instances, a vector can be a linearized vector. A linearizedvector can comprise a site-directed polypeptide and/or a nucleicacid-targeting nucleic acid. A linearized vector may not be a circularplasmid. A linearized vector can comprise a double-stranded break. Alinearized vector may comprise a sequence encoding a fluorescent protein(e.g., orange fluorescent protein (OFP)). A linearized vector maycomprise a sequence encoding an antigen (e.g., CD4). A linearized vectorcan be linearized (e.g., cut) in a region of the vector encoding partsof the nucleic acid-targeting nucleic acid. For example a linearizedvector can be linearized (e.g., cut) in a region of the nucleicacid-targeting nucleic acid 5′ to the crRNA portion of the nucleicacid-targeting nucleic acid. A linearized vector can be linearized(e.g., cut) in a region of the nucleic acid-targeting nucleic acid 3′ tothe spacer extension sequence of the nucleic acid-targeting nucleicacid. A linearized vector can be linearized (e.g., cut) in a region ofthe nucleic acid-targeting nucleic acid encoding the crRNA sequence ofthe nucleic acid-targeting nucleic acid. In some instances, a linearizedvector or a closed supercoiled vector comprises a sequence encoding asite-directed polypeptide (e.g., Cas9), a promoter driving expression ofthe sequence encoding the site-directed polypeptide (e.g., CMVpromoter), a sequence encoding a linker (e.g., 2A), a sequence encodinga marker (e.g., CD4 or OFP), a sequence encoding portion of a nucleicacid-targeting nucleic acid, a promoter driving expression of thesequence encoding a portion of the nucleic acid-targeting nucleic acid,and a sequence encoding a selectable marker (e.g., ampicillin), or anycombination thereof.

A vector can comprise a transcription and/or translation controlelement. Depending on the host/vector system utilized, any of a numberof suitable transcription and translation control elements, includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, etc. may be used in the expression vector.

In some embodiments, a nucleotide sequence encoding a nucleicacid-targeting nucleic acid of the disclosure, a site-directedpolypeptide of the disclosure, an effector protein, a multiplexedgenetic targeting agent, a donor polynucleotide, a tandem fusionprotein, a reporter element, a genetic element of interest, a componentof a split system and/or any nucleic acid or proteinaceous moleculenecessary to carry out the embodiments of the methods of the disclosurecan be operably linked to a control element (e.g., a transcriptionalcontrol element), such as a promoter. The transcriptional controlelement may be functional in a eukaryotic cell, (e.g., a mammaliancell), a prokaryotic cell (e.g., bacterial or archaeal cell). In someembodiments, a nucleotide sequence encoding a nucleic acid-targetingnucleic acid of the disclosure, a site-directed polypeptide of thedisclosure, an effector protein, a multiplexed genetic targeting agent,a donor polynucleotide, a tandem fusion protein, a reporter element, agenetic element of interest, a component of a split system and/or anynucleic acid or proteinaceous molecule necessary to carry out theembodiments of the methods of the disclosure can be operably linked tomultiple control elements. Operable linkage to multiple control elementscan allow expression of the nucleotide sequence encoding a nucleicacid-targeting nucleic acid of the disclosure, a site-directedpolypeptide of the disclosure, an effector protein, a multiplexedgenetic targeting agent, a donor polynucleotide, a tandem fusionprotein, a reporter element, a genetic element of interest, a componentof a split system and/or any nucleic acid or proteinaceous moleculenecessary to carry out the embodiments of the methods of the disclosurein either prokaryotic or eukaryotic cells.

Non-limiting examples of suitable eukaryotic promoters (i.e. promotersfunctional in a eukaryotic cell) can include those from cytomegalovirus(CMV) immediate early, herpes simplex virus (HSV) thymidine kinase,early and late SV40, long terminal repeats (LTRs) from retrovirus, humanelongation factor-1 promoter (EF1), a hybrid construct comprising thecytomegalovirus (CMV) enhancer fused to the chicken beta-active promoter(CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1locus promoter (PGK) and mouse metallothionein-I. The promoter can be afungi promoter. The promoter can be a plant promoter. A database ofplant promoteres can be found (e.g., PlantProm). The expression vectormay also contain a ribosome binding site for translation initiation anda transcription terminator. The expression vector may also includeappropriate sequences for amplifying expression. The expression vectormay also include nucleotide sequences encoding non-native tags (e.g.,6×His tag, hemagglutinin tag, green fluorescent protein, etc.) that arefused to the site-directed polypeptide, thus resulting in a fusionprotein.

In some embodiments, a nucleotide sequence or sequences encoding anucleic acid-targeting nucleic acid of the disclosure, a site-directedpolypeptide of the disclosure, an effector protein, a multiplexedgenetic targeting agent, a donor polynucleotide, a tandem fusionprotein, a reporter element, a genetic element of interest, a componentof a split system and/or any nucleic acid or proteinaceous moleculenecessary to carry out the embodiments of the methods of the disclosurecan be operably linked to an inducible promoter (e.g., heat shockpromoter, tetracycline-regulated promoter, steroid-regulated promoter,metal-regulated promoter, estrogen receptor-regulated promoter, etc.).In some embodiments, a nucleotide sequence encoding a nucleicacid-targeting nucleic acid of the disclosure, a site-directedpolypeptide of the disclosure, an effector protein, a multiplexedgenetic targeting agent, a donor polynucleotide, a tandem fusionprotein, a reporter element, a genetic element of interest, a componentof a split system and/or any nucleic acid or proteinaceous moleculenecessary to carry out the embodiments of the methods of the disclosurecan be operably linked to a constitutive promoter (e.g., CMV promoter,UBC promoter). In some embodiments, the nucleotide sequence can beoperably linked to a spatially restricted and/or temporally restrictedpromoter (e.g., a tissue specific promoter, a cell type specificpromoter, etc.).

A nucleotide sequence or sequences encoding a nucleic acid-targetingnucleic acid of the disclosure, a site-directed polypeptide of thedisclosure, an effector protein, a multiplexed genetic targeting agent,a donor polynucleotide, a tandem fusion protein, a reporter element, agenetic element of interest, a component of a split system and/or anynucleic acid or proteinaceous molecule necessary to carry out theembodiments of the methods of the disclosure can be packaged into or onthe surface of biological compartments for delivery to cells. Biologicalcompartments can include, but are not limited to, viruses (lentivirus,adenovirus), nanospheres, liposomes, quantum dots, nanoparticles,polyethylene glycol particles, hydrogels, and micelles.

Introduction of the complexes, polypeptides, and nucleic acids of thedisclosure into cells can occur by viral or bacteriophage infection,transfection, conjugation, protoplast fusion, lipofection,electroporation, calcium phosphate precipitation, polyethyleneimine(PEI)-mediated transfection, DEAE-dextran mediated transfection,liposome-mediated transfection, particle gun technology, calciumphosphate precipitation, direct micro-injection, nanoparticle-mediatednucleic acid delivery, and the like.

Transgenic Cells and Organisms

The disclosure provides for transgenic cells and organisms. The nucleicacid of a genetically modified host cell and/or transgenic organism canbe targeted for genome engineering.

Exemplary cells that can be used to generate transgenic cells accordingto the methods of the disclosure can include, but are not limited to,HeLa cell, Chinese Hamster Ovary cell, 293-T cell, a pheochromocytoma, aneuroblastomas fibroblast, a rhabdomyosarcoma, a dorsal root ganglioncell, a NSO cell, Tobacco BY-2, CV-I (ATCC CCL 70), COS-I (ATCC CRL1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC, CCL 61), 3T3 (ATCC CCL 92),NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C 1271 (ATCC CRL 1616),BS-C-I (ATCC CCL 26), MRC-5 (ATCC CCL 171), L-cells, HEK-293 (ATCC CRL1573) and PC 12 (ATCC CRL-1721), HEK293T (ATCC CRL-11268), RBL (ATCCCRL-1378), SH-SY5Y (ATCC CRL-2266), MDCK (ATCC CCL-34), SJ-RH30 (ATCCCRL-2061.), HepG2 (ATCC ND7/23 (ECACC 92090903), CHO (ECACC 85050302),Vera (ATCC CCL 81), Caco-2 (ATCC HTB 37), K562 (ATCC CCL 243), Jurkat(ATCC TIB-152), Per.C{dot over (o)}, Huvec (ATCC Human Primary PCS100-010, Mouse CRL 2514, CRL 2515, CRL 2516), HuH-7D12 (ECACC 01042712),293 (ATCC CRL 10852), A549 (ATCC CCL 185), IMR-90 (ATCC CCL 186), MCF-7(ATC HTB-22), U-2 OS (ATCC HTB-96), and T84 (ATCC CCL 248), or any cellavailable at American Type Culture Collection (ATCC), or any combinationthereof.

Organisms that can be transgenic can include bacteria, archaea,single-cell eukaryotes, plants, algae, fungi (e.g., yeast),invertebrates (e.g., fruit fly, cnidarian, echinoderm, nematode, etc),vertebrates (e.g., fish, amphibian, reptile, bird, mammal), mammalsmammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, anon-human primate, a human, etc.), etc.

Transgenic organisms can comprise genetically modified cells. Transgenicorganisms and/or genetically modified cells can comprise organismsand/or cells that have been genetically modified with an exogenousnucleic acid comprising a nucleotide sequence encoding nucleicacid-targeting nucleic acid of the disclosure, an effector protein,and/or a site-directed polypeptide, or any combination thereof.

A genetically modified cell can comprise an exogenous site-directedpolypeptide and/or an exogenous nucleic acid comprising a nucleotidesequence encoding a site-directed polypeptide. Expression of thesite-directed polypeptide in the cell may take 0.1, 0.2, 0.5, 1, 2, 3,4, 5, 6, or more days. Cells, introduced with the site-directedpolypeptide, may be grown for 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or even more days before the cells can be removed from cell cultureand/or host organism.

Subjects

The disclosure provides for performing the methods of the disclosure ina subject. A subject can be a human. A subject can be a mammal (e.g.,rat, mouse, cow, dog, pig, sheep, horse). A subject can be a vetebrateor an invertebrate. A subject can be a laboratory animal. A subject canbe a patient. A subject can be suffering from a disease. A subject candisplay symptoms of a disease. A subject may not display symptoms of adisease, but still have a disease. A subject can be under medical careof a caregiver (e.g., the subject is hospitalized and is treated by aphysician). A subject can be a plant or a crop.

Kits

The present disclosure provides kits for carrying out the methods of thedisclosure. A kit can include one or more of: A nucleic acid-targetingnucleic acid of the disclosure, a polynucleotide encoding a nucleicacid-targeting nucleic acid, a site-directed polypeptide of thedisclosure, a polynucleotide encoding a site-directed polypeptide, aneffector protein, a polynucleotide encoding an effector protein, amultiplexed genetic targeting agent, a polynucleotide encoding amultiplexed genetic targeting agent, a donor polynucleotide, a tandemfusion protein, a polynucleotide encoding a tandem fusion protein, areporter element, a genetic element of interest, a component of a splitsystem and/or any nucleic acid or proteinaceous molecule necessary tocarry out the embodiments of the methods of the disclosure, or anycombination thereof.

A nucleic acid-targeting nucleic acid of the disclosure, apolynucleotide encoding a nucleic acid-targeting nucleic acid, asite-directed polypeptide of the disclosure, a polynucleotide encoding asite-directed polypeptide, an effector protein, a polynucleotideencoding an effector protein, a multiplexed genetic targeting agent, apolynucleotide encoding a multiplexed genetic targeting agent, a donorpolynucleotide, a tandem fusion protein, a polynucleotide encoding atandem fusion protein, a reporter element, a genetic element ofinterest, a component of a split system and/or any nucleic acid orproteinaceous molecule necessary to carry out the embodiments of themethods of the disclosure are described in detail above.

A kit can comprise: (1) a vector comprising a nucleotide sequenceencoding a nucleic acid-targeting nucleic acid, and (2) a vectorcomprising a nucleotide sequence encoding the site-directed polypeptideand (2) a reagent for reconstitution and/or dilution of the vectors.

A kit can comprise: (1) a vector comprising (i) a nucleotide sequenceencoding a nucleic acid-targeting nucleic acid, and (ii) a nucleotidesequence encoding the site-directed polypeptide and (2) a reagent forreconstitution and/or dilution of the vector.

A kit can comprise: (1) a vector comprising a nucleotide sequenceencoding a nucleic acid-targeting nucleic acid, (2) a vector comprisinga nucleotide sequence encoding the site-directed polypeptide, (3) avector comprising a nucleotide sequence encoding an effector protein, amultiplexed genetic targeting agent, a donor polynucleotide, a tandemfusion protein, a reporter element, a genetic element of interest, acomponent of a split system and/or any nucleic acid or proteinaceousmolecule necessary to carry out the embodiments of the methods of thedisclosure, and (4) a reagent for reconstitution and/or dilution of thevectors.

A kit can comprise: (1) a vector comprising (i) a nucleotide sequenceencoding a nucleic acid-targeting nucleic acid, (ii) a nucleotidesequence encoding the site-directed polypeptide, (2) a vector comprisinga nucleotide sequence encoding an effector protein, a multiplexedgenetic targeting agent, a donor polynucleotide, a tandem fusionprotein, a reporter element, a genetic element of interest, a componentof a split system and/or any nucleic acid or proteinaceous moleculenecessary to carry out the embodiments of the methods of the disclosure,and (3) a reagent for reconstitution and/or dilution of the recombinantexpression vectors.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide can comprise an amino acid sequence comprising at least 15%amino acid identity to a Cas9 from S. pyogenes, and two nucleic acidcleaving domains (i.e., an HNH domain and a RuvC domain); and adouble-guide nucleic acid-targeting nucleic acid (and/or a nucleic acidencoding the same) comprising 1) a first nucleic acid moleculecomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; and aminimum CRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6 contiguous nucleotidesand wherein the minimum CRISPR repeat has a length from 5-30nucleotides; and 2) a second nucleic acid molecule of the double-guidenucleic acid-targeting nucleic acid can comprise a minimum tracrRNAsequence comprising at least 60% identity to a tracrRNA from aprokaryote (e.g., S. pyogenes) or phage over 6 contiguous nucleotidesand wherein the minimum tracrRNA sequence has a length from 5-30nucleotides; a 3′ tracrRNA that comprises at least 60% identity to atracrRNA from a bacterium (e.g., S. pyogenes) over 6 contiguousnucleotides and wherein the 3′ tracrRNA comprises a length from 10-20nucleotides, and comprises a duplexed region; and/or a tracrRNAextension comprising 10-5000 nucleotides in length, or any combinationthereof.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide can comprise an amino acid sequence comprising at least 15%amino acid identity to a Cas9 from S. pyogenes, and two nucleic acidcleaving domains (i.e., an HNH domain and a RuvC domain); and, adouble-guide nucleic acid-targeting nucleic acid (and/or a nucleic acidencoding the same) comprising 1) a first nucleic acid moleculecomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a minimumCRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6 contiguous nucleotidesand wherein the minimum CRISPR repeat has a length from 5-30nucleotides, and at least 3 unpaired nucleotides of a bulge; and 2) asecond nucleic acid molecule of the double-guide nucleic acid-targetingnucleic acid can comprise a minimum tracrRNA sequence comprising atleast 60% identity to a tracrRNA from a prokaryote (e.g., S. pyogenes)or phage over 6 contiguous nucleotides and wherein the minimum tracrRNAsequence has a length from 5-30 nucleotides and at least 1 unpairednucleotide of a bulge, wherein the 1 unpaired nucleotide of the bulge islocated in the same bulge as the 3 unpaired nucleotides of the minimumCRISPR repeat; a 3′ tracrRNA that comprises at least 60% identity to atracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6contiguous nucleotides and wherein the 3′ tracrRNA comprises a lengthfrom 10-20 nucleotides, and comprises a duplexed region; a P-domain thatstarts from 1-5 nucleotides downstream of the duplex comprising theminimum CRISPR repeat and the minimum tracrRNA, comprises 1-10nucleotides, comprises a sequence that can hybridize to a protospaceradjacent motif in a target nucleic acid, can form a hairpin, and islocated in the 3′ tracrRNA region; and/or a tracrRNA extensioncomprising 10-5000 nucleotides in length, or any combination thereof.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide can comprise an amino acid sequence comprising at least 15%amino acid identity to a Cas9 from S. pyogenes and, two nucleic acidcleaving domains (i.e., an HNH domain and a RuvC domain); and, a nucleicacid-targeting nucleic acid (and/or a nucleic acid encoding the same)comprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a minimumCRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguousnucleotides and wherein the minimum CRISPR repeat has a length from 5-30nucleotides; a minimum tracrRNA sequence comprising at least 60%identity to a tracrRNA from a bacterium (e.g., S. pyogenes) over 6, 7,or 8 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6, 7, or 8 contiguous nucleotides and wherein the 3′ tracrRNAcomprises a length from 10-20 nucleotides, and comprises a duplexedregion; and/or a tracrRNA extension comprising 10-5000 nucleotides inlength, or any combination thereof. This nucleic acid-targeting nucleicacid can be referred to as a single guide nucleic acid-targeting nucleicacid.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide can comprise an amino acid sequence comprising at least 15%amino acid identity to a Cas9 from S. pyogenes, and two nucleic acidcleaving domains (i.e., an HNH domain and a RuvC domain); and, a nucleicacid-targeting nucleic acid (and/or a nucleic acid encoding the same)can comprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a duplexcomprising 1) a minimum CRISPR repeat comprising at least 60% identityto a crRNA from a prokaryote (e.g., S. pyogenes) or phage over 6contiguous nucleotides and wherein the minimum CRISPR repeat has alength from 5-30 nucleotides, 2) a minimum tracrRNA sequence comprisingat least 60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)over 6 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides, and 3) a bulge wherein the bulgecomprises at least 3 unpaired nucleotides on the minimum CRISPR repeatstrand of the duplex and at least 1 unpaired nucleotide on the minimumtracrRNA sequence strand of the duplex; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6 contiguous nucleotides, wherein the 3′ tracrRNA comprises alength from 10-20 nucleotides and comprises a duplexed region; aP-domain that starts from 1-5 nucleotides downstream of the duplexcomprising the minimum CRISPR repeat and the minimum tracrRNA, comprises1-10 nucleotides, comprises a sequence that can hybridize to aprotospacer adjacent motif in a target nucleic acid, can form a hairpin,and is located in the 3′ tracrRNA region; and/or a tracrRNA extensioncomprising 10-5000 nucleotides in length, or any combination thereof.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide comprises an amino acid sequence comprising at least 15%amino acid identity to a Cas9 from S. pyogenes, two nucleic acidcleaving domains (i.e., an HNH domain and a RuvC domain), and a linkerlinking the site-directed polypeptide to a non-native sequence; and adouble-guide nucleic acid-targeting nucleic acid (and/or a nucleic acidencoding the same) comprising 1) a first nucleic acid moleculecomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; and aminimum CRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6 contiguous nucleotidesand wherein the minimum CRISPR repeat has a length from 5-30nucleotides; and 2) a second nucleic acid molecule of the double-guidenucleic acid-targeting nucleic acid can comprise a minimum tracrRNAsequence comprising at least 60% identity to a tracrRNA from aprokaryote (e.g., S. pyogenes) or phage over 6 contiguous nucleotidesand wherein the minimum tracrRNA sequence has a length from 5-30nucleotides; a 3′ tracrRNA that comprises at least 60% identity to atracrRNA from a bacterium (e.g., S. pyogenes) over 6 contiguousnucleotides and wherein the 3′ tracrRNA comprises a length from 10-20nucleotides, and comprises a duplexed region; and/or a tracrRNAextension comprising 10-5000 nucleotides in length, or any combinationthereof.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide comprises an amino acid sequence comprising at least 15%amino acid identity to a Cas9 from S. pyogenes, two nucleic acidcleaving domains (i.e., an HNH domain and a RuvC domain), and a linkerlinking the site-directed polypeptide to a non-native sequence; and adouble-guide nucleic acid-targeting nucleic acid (and/or a nucleic acidencoding the same) comprising 1) a first nucleic acid moleculecomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a minimumCRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6 contiguous nucleotidesand wherein the minimum CRISPR repeat has a length from 5-30nucleotides, and at least 3 unpaired nucleotides of a bulge; and 2) asecond nucleic acid molecule of the double-guide nucleic acid-targetingnucleic acid can comprise a minimum tracrRNA sequence comprising atleast 60% identity to a tracrRNA from a prokaryote (e.g., S. pyogenes)or phage over 6 contiguous nucleotides and wherein the minimum tracrRNAsequence has a length from 5-30 nucleotides and at least 1 unpairednucleotide of a bulge, wherein the 1 unpaired nucleotide of the bulge islocated in the same bulge as the 3 unpaired nucleotides of the minimumCRISPR repeat; a 3′ tracrRNA that comprises at least 60% identity to atracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6contiguous nucleotides and wherein the 3′ tracrRNA comprises a lengthfrom 10-20 nucleotides, and comprises a duplexed region; a P-domain thatstarts from 1-5 nucleotides downstream of the duplex comprising theminimum CRISPR repeat and the minimum tracrRNA, comprises 1-10nucleotides, comprises a sequence that can hybridize to a protospaceradjacent motif in a target nucleic acid, can form a hairpin, and islocated in the 3′ tracrRNA region; and/or a tracrRNA extensioncomprising 10-5000 nucleotides in length, or any combination thereof.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide comprises an amino acid sequence comprising at least 15%amino acid identity to a Cas9 from S. pyogenes, two nucleic acidcleaving domains (i.e., an HNH domain and a RuvC domain), and a linkerlinking the site-directed polypeptide to a non-native sequence; and anucleic acid-targeting nucleic acid (and/or a nucleic acid encoding thesame) comprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a minimumCRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguousnucleotides and wherein the minimum CRISPR repeat has a length from 5-30nucleotides; a minimum tracrRNA sequence comprising at least 60%identity to a tracrRNA from a bacterium (e.g., S. pyogenes) over 6, 7,or 8 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6, 7, or 8 contiguous nucleotides and wherein the 3′ tracrRNAcomprises a length from 10-20 nucleotides, and comprises a duplexedregion; and/or a tracrRNA extension comprising 10-5000 nucleotides inlength, or any combination thereof. This nucleic acid-targeting nucleicacid can be referred to as a single guide nucleic acid-targeting nucleicacid.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide comprises an amino acid sequence comprising at least 15%amino acid identity to a Cas9 from S. pyogenes, two nucleic acidcleaving domains (i.e., an HNH domain and a RuvC domain), and a linkerlinking the site-directed polypeptide to a non-native sequence; andnucleic acid-targeting nucleic acid (and/or a nucleic acid encoding thesame) comprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a duplexcomprising 1) a minimum CRISPR repeat comprising at least 60% identityto a crRNA from a prokaryote (e.g., S. pyogenes) or phage over 6contiguous nucleotides and wherein the minimum CRISPR repeat has alength from 5-30 nucleotides, 2) a minimum tracrRNA sequence comprisingat least 60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)over 6 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides, and 3) a bulge wherein the bulgecomprises at least 3 unpaired nucleotides on the minimum CRISPR repeatstrand of the duplex and at least 1 unpaired nucleotide on the minimumtracrRNA sequence strand of the duplex; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6 contiguous nucleotides, wherein the 3′ tracrRNA comprises alength from 10-20 nucleotides and comprises a duplexed region; aP-domain that starts from 1-5 nucleotides downstream of the duplexcomprising the minimum CRISPR repeat and the minimum tracrRNA, comprises1-10 nucleotides, comprises a sequence that can hybridize to aprotospacer adjacent motif in a target nucleic acid, can form a hairpin,and is located in the 3′ tracrRNA region; and/or a tracrRNA extensioncomprising 10-5000 nucleotides in length, or any combination thereof.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide comprises at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid cleaving domains (i.e., an HNH domain and aRuvC domain), wherein the site-directed polypeptide comprises a mutationin one or both of the nucleic acid cleaving domains that reduces thecleaving activity of the nuclease domains by at least 50%; and adouble-guide nucleic acid-targeting nucleic acid (and/or a nucleic acidencoding the same) comprising 1) a first nucleic acid moleculecomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; and aminimum CRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6 contiguous nucleotidesand wherein the minimum CRISPR repeat has a length from 5-30nucleotides; and 2) a second nucleic acid molecule of the double-guidenucleic acid-targeting nucleic acid can comprise a minimum tracrRNAsequence comprising at least 60% identity to a tracrRNA from aprokaryote (e.g., S. pyogenes) or phage over 6 contiguous nucleotidesand wherein the minimum tracrRNA sequence has a length from 5-30nucleotides; a 3′ tracrRNA that comprises at least 60% identity to atracrRNA from a bacterium (e.g., S. pyogenes) over 6 contiguousnucleotides and wherein the 3′ tracrRNA comprises a length from 10-20nucleotides, and comprises a duplexed region; and/or a tracrRNAextension comprising 10-5000 nucleotides in length, or any combinationthereof.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide comprises at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid cleaving domains (i.e., an HNH domain and aRuvC domain), wherein the site-directed polypeptide comprises a mutationin one or both of the nucleic acid cleaving domains that reduces thecleaving activity of the nuclease domains by at least 50%; and adouble-guide nucleic acid-targeting nucleic acid (and/or a nucleic acidencoding the same) comprising 1) a first nucleic acid moleculecomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a minimumCRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6 contiguous nucleotidesand wherein the minimum CRISPR repeat has a length from 5-30nucleotides, and at least 3 unpaired nucleotides of a bulge; and 2) asecond nucleic acid molecule of the double-guide nucleic acid-targetingnucleic acid can comprise a minimum tracrRNA sequence comprising atleast 60% identity to a tracrRNA from a prokaryote (e.g., S. pyogenes)or phage over 6 contiguous nucleotides and wherein the minimum tracrRNAsequence has a length from 5-30 nucleotides and at least 1 unpairednucleotide of a bulge, wherein the 1 unpaired nucleotide of the bulge islocated in the same bulge as the 3 unpaired nucleotides of the minimumCRISPR repeat; a 3′ tracrRNA that comprises at least 60% identity to atracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6contiguous nucleotides and wherein the 3′ tracrRNA comprises a lengthfrom 10-20 nucleotides, and comprises a duplexed region; a P-domain thatstarts from 1-5 nucleotides downstream of the duplex comprising theminimum CRISPR repeat and the minimum tracrRNA, comprises 1-10nucleotides, comprises a sequence that can hybridize to a protospaceradjacent motif in a target nucleic acid, can form a hairpin, and islocated in the 3′ tracrRNA region; and/or a tracrRNA extensioncomprising 10-5000 nucleotides in length, or any combination thereof.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide comprises at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid cleaving domains (i.e., an HNH domain and aRuvC domain), wherein the site-directed polypeptide comprises a mutationin one or both of the nucleic acid cleaving domains that reduces thecleaving activity of the nuclease domains by at least 50%; and nucleicacid-targeting nucleic acid (and/or a nucleic acid encoding the same)comprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a minimumCRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguousnucleotides and wherein the minimum CRISPR repeat has a length from 5-30nucleotides; a minimum tracrRNA sequence comprising at least 60%identity to a tracrRNA from a bacterium (e.g., S. pyogenes) over 6, 7,or 8 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6, 7, or 8 contiguous nucleotides and wherein the 3′ tracrRNAcomprises a length from 10-20 nucleotides, and comprises a duplexedregion; and/or a tracrRNA extension comprising 10-5000 nucleotides inlength, or any combination thereof. This nucleic acid-targeting nucleicacid can be referred to as a single guide nucleic acid-targeting nucleicacid.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide comprises at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid cleaving domains (i.e., an HNH domain and aRuvC domain), wherein the site-directed polypeptide comprises a mutationin one or both of the nucleic acid cleaving domains that reduces thecleaving activity of the nuclease domains by at least 50%; and nucleicacid-targeting nucleic acid (and/or a nucleic acid encoding the same)comprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a duplexcomprising 1) a minimum CRISPR repeat comprising at least 60% identityto a crRNA from a prokaryote (e.g., S. pyogenes) or phage over 6contiguous nucleotides and wherein the minimum CRISPR repeat has alength from 5-30 nucleotides, 2) a minimum tracrRNA sequence comprisingat least 60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)over 6 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides, and 3) a bulge wherein the bulgecomprises at least 3 unpaired nucleotides on the minimum CRISPR repeatstrand of the duplex and at least 1 unpaired nucleotide on the minimumtracrRNA sequence strand of the duplex; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6 contiguous nucleotides, wherein the 3′ tracrRNA comprises alength from 10-20 nucleotides and comprises a duplexed region; aP-domain that starts from 1-5 nucleotides downstream of the duplexcomprising the minimum CRISPR repeat and the minimum tracrRNA, comprises1-10 nucleotides, comprises a sequence that can hybridize to aprotospacer adjacent motif in a target nucleic acid, can form a hairpin,and is located in the 3′ tracrRNA region; and/or a tracrRNA extensioncomprising 10-5000 nucleotides in length, or any combination thereof.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide comprises at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid cleaving domains (i.e., an HNH domain and aRuvC domain), wherein the site-directed polypeptide comprises a mutationin one or both of the nucleic acid cleaving domains that reduces thecleaving activity of the nuclease domains by at least 50%; and adouble-guide nucleic acid-targeting nucleic acid (and/or a nucleic acidencoding the same) comprising 1) a first nucleic acid moleculecomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; and aminimum CRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6 contiguous nucleotidesand wherein the minimum CRISPR repeat has a length from 5-30nucleotides; and 2) a second nucleic acid molecule of the double-guidenucleic acid-targeting nucleic acid can comprise a minimum tracrRNAsequence comprising at least 60% identity to a tracrRNA from aprokaryote (e.g., S. pyogenes) or phage over 6 contiguous nucleotidesand wherein the minimum tracrRNA sequence has a length from 5-30nucleotides; a 3′ tracrRNA that comprises at least 60% identity to atracrRNA from a bacterium (e.g., S. pyogenes) over 6 contiguousnucleotides and wherein the 3′ tracrRNA comprises a length from 10-20nucleotides, and comprises a duplexed region; and/or a tracrRNAextension comprising 10-5000 nucleotides in length, or any combinationthereof.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide comprises at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid cleaving domains (i.e., an HNH domain and aRuvC domain), wherein the site-directed polypeptide comprises a mutationin one or both of the nucleic acid cleaving domains that reduces thecleaving activity of the nuclease domains by at least 50%; and adouble-guide nucleic acid-targeting nucleic acid (and/or a nucleic acidencoding the same) comprising 1) a first nucleic acid moleculecomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a minimumCRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6 contiguous nucleotidesand wherein the minimum CRISPR repeat has a length from 5-30nucleotides, and at least 3 unpaired nucleotides of a bulge; and 2) asecond nucleic acid molecule of the double-guide nucleic acid-targetingnucleic acid can comprise a minimum tracrRNA sequence comprising atleast 60% identity to a tracrRNA from a prokaryote (e.g., S. pyogenes)or phage over 6 contiguous nucleotides and wherein the minimum tracrRNAsequence has a length from 5-30 nucleotides and at least 1 unpairednucleotide of a bulge, wherein the 1 unpaired nucleotide of the bulge islocated in the same bulge as the 3 unpaired nucleotides of the minimumCRISPR repeat; a 3′ tracrRNA that comprises at least 60% identity to atracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6contiguous nucleotides and wherein the 3′ tracrRNA comprises a lengthfrom 10-20 nucleotides, and comprises a duplexed region; a P-domain thatstarts from 1-5 nucleotides downstream of the duplex comprising theminimum CRISPR repeat and the minimum tracrRNA, comprises 1-10nucleotides, comprises a sequence that can hybridize to a protospaceradjacent motif in a target nucleic acid, can form a hairpin, and islocated in the 3′ tracrRNA region; and/or a tracrRNA extensioncomprising 10-5000 nucleotides in length, or any combination thereof.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide comprises at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid cleaving domains (i.e., an HNH domain and aRuvC domain), wherein the site-directed polypeptide comprises a mutationin one or both of the nucleic acid cleaving domains that reduces thecleaving activity of the nuclease domains by at least 50%; and nucleicacid-targeting nucleic acid (and/or a nucleic acid encoding the same)comprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a minimumCRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguousnucleotides and wherein the minimum CRISPR repeat has a length from 5-30nucleotides; a minimum tracrRNA sequence comprising at least 60%identity to a tracrRNA from a bacterium (e.g., S. pyogenes) over 6, 7,or 8 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6, 7, or 8 contiguous nucleotides and wherein the 3′ tracrRNAcomprises a length from 10-20 nucleotides, and comprises a duplexedregion; and/or a tracrRNA extension comprising 10-5000 nucleotides inlength, or any combination thereof. This nucleic acid-targeting nucleicacid can be referred to as a single guide nucleic acid-targeting nucleicacid.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide comprises at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid cleaving domains (i.e., an HNH domain and aRuvC domain), wherein the site-directed polypeptide comprises a mutationin one or both of the nucleic acid cleaving domains that reduces thecleaving activity of the nuclease domains by at least 50%; and a nucleicacid-targeting nucleic acid (and/or a nucleic acid encoding the same)comprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a duplexcomprising 1) a minimum CRISPR repeat comprising at least 60% identityto a crRNA from a prokaryote (e.g., S. pyogenes) or phage over 6contiguous nucleotides and wherein the minimum CRISPR repeat has alength from 5-30 nucleotides, 2) a minimum tracrRNA sequence comprisingat least 60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)over 6 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides, and 3) a bulge wherein the bulgecomprises at least 3 unpaired nucleotides on the minimum CRISPR repeatstrand of the duplex and at least 1 unpaired nucleotide on the minimumtracrRNA sequence strand of the duplex; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6 contiguous nucleotides, wherein the 3′ tracrRNA comprises alength from 10-20 nucleotides and comprises a duplexed region; aP-domain that starts from 1-5 nucleotides downstream of the duplexcomprising the minimum CRISPR repeat and the minimum tracrRNA, comprises1-10 nucleotides, comprises a sequence that can hybridize to aprotospacer adjacent motif in a target nucleic acid, can form a hairpin,and is located in the 3′ tracrRNA region; and/or a tracrRNA extensioncomprising 10-5000 nucleotides in length, or any combination thereof.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide comprises at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid cleaving domains (i.e., an HNH domain and aRuvC domain), wherein the site-directed polypeptide comprises a mutationin one or both of the nucleic acid cleaving domains that reduces thecleaving activity of the nuclease domains by at least 50%; and adouble-guide nucleic acid-targeting nucleic acid (and/or a nucleic acidencoding the same) comprising 1) a first nucleic acid moleculecomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; and aminimum CRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6 contiguous nucleotidesand wherein the minimum CRISPR repeat has a length from 5-30nucleotides; and 2) a second nucleic acid molecule of the double-guidenucleic acid-targeting nucleic acid can comprise a minimum tracrRNAsequence comprising at least 60% identity to a tracrRNA from aprokaryote (e.g., S. pyogenes) or phage over 6 contiguous nucleotidesand wherein the minimum tracrRNA sequence has a length from 5-30nucleotides; a 3′ tracrRNA that comprises at least 60% identity to atracrRNA from a bacterium (e.g., S. pyogenes) over 6 contiguousnucleotides and wherein the 3′ tracrRNA comprises a length from 10-20nucleotides, and comprises a duplexed region; and/or a tracrRNAextension comprising 10-5000 nucleotides in length, or any combinationthereof.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide comprises at least 15% amino acid identity to a Cas9 from S.pyogenes, and two nucleic acid cleaving domains, wherein one or both ofthe nucleic acid cleaving domains comprise at least 50% amino acididentity to a nuclease domain from Cas9 from S. pyogenes; and adouble-guide nucleic acid-targeting nucleic acid (and/or a nucleic acidencoding the same) comprising: 1) a first nucleic acid moleculecomprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a minimumCRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6 contiguous nucleotidesand wherein the minimum CRISPR repeat has a length from 5-30nucleotides, and at least 3 unpaired nucleotides of a bulge; and 2) asecond nucleic acid molecule of the double-guide nucleic acid-targetingnucleic acid can comprise a minimum tracrRNA sequence comprising atleast 60% identity to a tracrRNA from a prokaryote (e.g., S. pyogenes)or phage over 6 contiguous nucleotides and wherein the minimum tracrRNAsequence has a length from 5-30 nucleotides and at least 1 unpairednucleotide of a bulge, wherein the 1 unpaired nucleotide of the bulge islocated in the same bulge as the 3 unpaired nucleotides of the minimumCRISPR repeat; a 3′ tracrRNA that comprises at least 60% identity to atracrRNA from a prokaryote (e.g., S. pyogenes) or phage over 6contiguous nucleotides and wherein the 3′ tracrRNA comprises a lengthfrom 10-20 nucleotides, and comprises a duplexed region; a P-domain thatstarts from 1-5 nucleotides downstream of the duplex comprising theminimum CRISPR repeat and the minimum tracrRNA, comprises 1-10nucleotides, comprises a sequence that can hybridize to a protospaceradjacent motif in a target nucleic acid, can form a hairpin, and islocated in the 3′ tracrRNA region; and/or a tracrRNA extensioncomprising 10-5000 nucleotides in length, or any combination thereof.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide comprises at least 15% amino acid identity to a Cas9 from S.pyogenes, and two nucleic acid cleaving domains, wherein one or both ofthe nucleic acid cleaving domains comprise at least 50% amino acididentity to a nuclease domain from Cas9 from S. pyogenes; and nucleicacid-targeting nucleic acid (and/or a nucleic acid encoding the same)comprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a minimumCRISPR repeat comprising at least 60% identity to a crRNA from aprokaryote (e.g., S. pyogenes) or phage over 6, 7, or 8 contiguousnucleotides and wherein the minimum CRISPR repeat has a length from 5-30nucleotides; a minimum tracrRNA sequence comprising at least 60%identity to a tracrRNA from a bacterium (e.g., S. pyogenes) over 6, 7,or 8 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6, 7, or 8 contiguous nucleotides and wherein the 3′ tracrRNAcomprises a length from 10-20 nucleotides, and comprises a duplexedregion; and/or a tracrRNA extension comprising 10-5000 nucleotides inlength, or any combination thereof. This nucleic acid-targeting nucleicacid can be referred to as a single guide nucleic acid-targeting nucleicacid.

In some instances, a kit can comprise a site-directed polypeptide(and/or a nucleic acid encoding the same), wherein the site-directedpolypeptide comprises at least 15% amino acid identity to a Cas9 from S.pyogenes, and two nucleic acid cleaving domains, wherein one or both ofthe nucleic acid cleaving domains comprise at least 50% amino acididentity to a nuclease domain from Cas9 from S. pyogenes; and a nucleicacid-targeting nucleic acid (and/or a nucleic acid encoding the same)comprising a spacer extension sequence from 10-5000 nucleotides inlength; a spacer sequence of 12-30 nucleotides in length, wherein thespacer is at least 50% complementary to a target nucleic acid; a duplexcomprising: 1) a minimum CRISPR repeat comprising at least 60% identityto a crRNA from a prokaryote (e.g., S. pyogenes) or phage over 6contiguous nucleotides and wherein the minimum CRISPR repeat has alength from 5-30 nucleotides, 2) a minimum tracrRNA sequence comprisingat least 60% identity to a tracrRNA from a bacterium (e.g., S. pyogenes)over 6 contiguous nucleotides and wherein the minimum tracrRNA sequencehas a length from 5-30 nucleotides, and 3) a bulge wherein the bulgecomprises at least 3 unpaired nucleotides on the minimum CRISPR repeatstrand of the duplex and at least 1 unpaired nucleotide on the minimumtracrRNA sequence strand of the duplex; a linker sequence that links theminimum CRISPR repeat and the minimum tracrRNA and comprises a lengthfrom 3-5000 nucleotides; a 3′ tracrRNA that comprises at least 60%identity to a tracrRNA from a prokaryote (e.g., S. pyogenes) or phageover 6 contiguous nucleotides, wherein the 3′ tracrRNA comprises alength from 10-20 nucleotides and comprises a duplexed region; aP-domain that starts from 1-5 nucleotides downstream of the duplexcomprising the minimum CRISPR repeat and the minimum tracrRNA, comprises1-10 nucleotides, comprises a sequence that can hybridize to aprotospacer adjacent motif in a target nucleic acid, can form a hairpin,and is located in the 3′ tracrRNA region; and/or a tracrRNA extensioncomprising 10-5000 nucleotides in length, or any combination thereof.

In some embodiments of any of the above kits, the kit can comprise asingle guide nucleic acid-targeting nucleic acid. In some embodiments ofany of the above kits, the kit can comprise a double guide nucleicacid-targeting nucleic acid. In some embodiments of any of the abovekits, the kit can comprise two or more double guide or single guidenucleic acid-targeting nucleic acids. In some embodiments, a vector mayencode for a nucleic acid targeting nucleic acid.

In some embodiments of any of the above kits, the kit can furthercomprise a donor polynucleotide, or a polynucleotide sequence encodingthe donor polynucleotide, to effect the desired genetic modification.Components of a kit can be in separate containers; or can be combined ina single container.

A kit described above further comprise one or more additional reagents,where such additional reagents can be selected from: a buffer, a bufferfor introducing the a polypeptide or polynucleotide item of the kit intoa cell, a wash buffer, a control reagent, a control vector, a controlRNA polynucleotide, a reagent for in vitro production of the polypeptidefrom DNA, adaptors for sequencing and the like. A buffer can be astabilization buffer, a reconstituting buffer, or a diluting buffer.

In some instances, a kit can comprise one or more additional reagentsspecific for plants and/or fungi. One or more additional reagents forplants and/or fungi can include, for example, soil, nutrients, plants,seeds, spores, Agrobacterium, T-DNA vector, and a pBINAR vector.

In addition to above-mentioned components, a kit can further includeinstructions for using the components of the kit to practice themethods. The instructions for practicing the methods are generallyrecorded on a suitable recording medium. For example, the instructionsmay be printed on a substrate, such as paper or plastic, etc. Theinstructions may be present in the kits as a package insert, in thelabeling of the container of the kit or components thereof (i.e.,associated with the packaging or subpackaging) etc. The instructions canbe present as an electronic storage data file present on a suitablecomputer readable storage medium, e.g. CD-ROM, diskette, flash drive,etc. In some instances, the actual instructions are not present in thekit, but means for obtaining the instructions from a remote source (e.g.via the Internet), can be provided. An example of this embodiment is akit that includes a web address where the instructions can be viewedand/or from which the instructions can be downloaded. As with theinstructions, this means for obtaining the instructions can be recordedon a suitable substrate.

In some embodiments, a kit can comprise a linearized vector. Alinearized vector can comprise a plasmid comprising a site-directedpolypeptide and/or a nucleic acid-targeting nucleic acid that islinearized (e.g., it is not circular). A linearized vector can be storedin a buffer comprising 10 mM Tris-HCl, pH 8.0 and 1 mM EDTA, pH 8.0. Akit can comprise about 20 microliters of the linearized CRISPR nucleasevector. In some embodiments, a kit can comprise one or more circularvectors.

In some embodiments a kit can comprise an oligonucleotide annealingbuffer. An oligonucleotide annealing buffer can be a buffer used toanneal DNA oligos together to generate a double-stranded DNA that encodea nucleic acid-targeting nucleic acid. A oligonucleotide annealingbuffer can be at least about, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or moreconcentrated than the concentration of use. An oligonucleotide annealingbuffer can be 10 times more concentrated than the concentration whenused. An oligonucleotide annealing buffer can comprise 100 mM Tris-HCl,pH 8.0, 10 mM EDTA, pH 8.0 and 1M NaCl. A kit can comprise 250microliters of the oligonucleotide annealing buffer.

A kit can comprise DNase-free water. A kit can comprise RNAse-freewater. A kit can comprise at least 1.5 milliliters of RNase-free and/orDNAse-free water.

A kit can comprise a ligation buffer. A ligation buffer can be used toligate oligonucleotides to the linearized CRISPR nuclease vector. Aligation buffer can be at least about, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10or more concentrated than the concentration of use. A ligation buffercan be 5 times as concentrated as the concentration of use. A 5×ligation buffer can comprise 250 mM Tris-HCl, pH 7.6, 50 mM MgCl₂, 5 mMATP, 5 mM DTT, and 25% (w/v) polyethylene glycol-8000. A kit cancomprise about 80 microliters of a ligation buffer.

A kit can comprise a DNA ligase. A DNA ligase can be used to ligate theoligonucleotides to the linearized CRISPR nuclease vector. A DNA ligasecan comprise 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM DTT, and 50% (v/v)glycerol. A kit can comprise 20 microliters of a DNA ligase.

A kit can comprise a sequencing primer. The sequencing primer can beused to sequence the vector once the oligonucleotides have been ligatedinto a linearized vector. A sequencing primer can be diluted inTris-EDTA buffer pH 8.0. A kit can comprise 20 microliters of asequencing primer.

A kit can comprise a control oligonucleotide. A control oligonucleotidecan be an oligonucleotide to be ligated into a linearized vector butdoes not encode for a nucleic acid-targeting nucleic acid. A controloligonucleotide can be diluted in 1× concentration of theoligonucleotide annealing buffer. A kit can comprise 10 microliters of acontrol oligonucleotide.

In some instances, a kit can comprise a linearized vector comprising asite-directed polypeptide and a nucleic acid-targeting nucleic acid, anoligonucleotide annealing buffer, DNAse/RNAse free water, a ligationbuffer, a ligase enzyme, a sequencing primer and a controloligonucleotide, or any combination thereof.

Pharmaceutical Compositions

Molecules, such as a nucleic acid-targeting nucleic acid of thedisclosure as described herein, a polynucleotide encoding a nucleicacid-targeting nucleic acid, a site-directed polypeptide of thedisclosure, a polynucleotide encoding a site-directed polypeptide, aneffector protein, a polynucleotide encoding an effector protein, amultiplexed genetic targeting agent, a polynucleotide encoding amultiplexed genetic targeting agent, a donor polynucleotide, a tandemfusion protein, a polynucleotide encoding a tandem fusion protein, areporter element, a genetic element of interest, a component of a splitsystem and/or any nucleic acid or proteinaceous molecule necessary tocarry out the embodiments of the methods of the disclosure, can beformulated in a pharmaceutical composition.

A pharmaceutical composition can comprise a combination of any moleculesdescribed herein with other chemical components, such as carriers,stabilizers, diluents, dispersing agents, suspending agents, thickeningagents, and/or excipients. The pharmaceutical composition can facilitateadministration of the molecule to an organism. Pharmaceuticalcompositions can be administered in therapeutically-effective amounts aspharmaceutical compositions by various forms and routes including, forexample, intravenous, subcutaneous, intramuscular, oral, rectal,aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, optic,nasal, and topical administration.

A pharmaceutical composition can be administered in a local or systemicmanner, for example, via injection of the molecule directly into anorgan, optionally in a depot or sustained release formulation.Pharmaceutical compositions can be provided in the form of a rapidrelease formulation, in the form of an extended release formulation, orin the form of an intermediate release formulation. A rapid release formcan provide an immediate release. An extended release formulation canprovide a controlled release or a sustained delayed release.

For oral administration, pharmaceutical compositions can be formulatedreadily by combining the molecules with pharmaceutically-acceptablecarriers or excipients. Such carriers can be used to formulate tablets,powders, pills, dragees, capsules, liquids, gels, syrups, elixirs,slurries, suspensions and the like, for oral ingestion by a subject.

Pharmaceutical preparations for oral use can be obtained by mixing oneor more solid excipient with one or more of the molecules describedherein, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Cores can be provided with suitablecoatings. For this purpose, concentrated sugar solutions can be used,which can contain an excipient such as gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments can be added to the tablets or drageecoatings, for example, for identification or to characterize differentcombinations of active compound doses.

Pharmaceutical preparations which can be used orally can includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. In someembodiments, the capsule comprises a hard gelatin capsule comprising oneor more of pharmaceutical, bovine, and plant gelatins. A gelatin can bealkaline-processed. The push-fit capsules can comprise the activeingredients in admixture with filler such as lactose, binders such asstarches, and/or lubricants such as talc or magnesium stearate and,stabilizers. In soft capsules, the molecule can be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. Stabilizers can be added. All formulationsfor oral administration are provided in dosages suitable for suchadministration.

For buccal or sublingual administration, the compositions can betablets, lozenges, or gels.

Parental injections can be formulated for bolus injection or continuousinfusion. The pharmaceutical compositions can be in a form suitable forparenteral injection as a sterile suspension, solution or emulsion inoily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Pharmaceuticalformulations for parenteral administration can include aqueous solutionsof the active compounds in water-soluble form.

Suspensions of molecules can be prepared as oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. Aqueous injection suspensions can containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. The suspension canalso contain suitable stabilizers or agents which increase thesolubility of the molecules to allow for the preparation of highlyconcentrated solutions. Alternatively, the active ingredient can be inpowder form for constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

The active compounds can be administered topically and can be formulatedinto a variety of topically administrable compositions, such assolutions, suspensions, lotions, gels, pastes, medicated sticks, balms,creams, and ointments. Such pharmaceutical compositions can comprisesolubilizers, stabilizers, tonicity enhancing agents, buffers andpreservatives.

Formulations suitable for transdermal administration of the moleculescan employ transdermal delivery devices and transdermal deliverypatches, and can be lipophilic emulsions or buffered aqueous solutions,dissolved and/or dispersed in a polymer or an adhesive. Such patches canbe constructed for continuous, pulsatile, or on demand delivery ofmolecules. Transdermal delivery can be accomplished by means ofiontophoretic patches and the like. Additionally, transdermal patchescan provide controlled delivery. The rate of absorption can be slowed byusing rate-controlling membranes or by trapping the compound within apolymer matrix or gel. Conversely, absorption enhancers can be used toincrease absorption. An absorption enhancer or carrier can includeabsorbable pharmaceutically acceptable solvents to assist passagethrough the skin. For example, transdermal devices can be in the form ofa bandage comprising a backing member, a reservoir containing compoundsand carriers, a rate controlling barrier to deliver the compounds to theskin of the subject at a controlled and predetermined rate over aprolonged period of time, and adhesives to secure the device to theskin.

For administration by inhalation, the molecule can be in a form as anaerosol, a mist, or a powder. Pharmaceutical compositions can bedelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebuliser, with the use of a suitable propellant, forexample, dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator can beformulated containing a powder mix of the compounds and a suitablepowder base such as lactose or starch.

The molecules can also be formulated in rectal compositions such asenemas, rectal gels, rectal foams, rectal aerosols, suppositories, jellysuppositories, or retention enemas, containing conventional suppositorybases such as cocoa butter or other glycerides, as well as syntheticpolymers such as polyvinylpyrrolidone and PEG. In suppository forms ofthe compositions, a low-melting wax such as a mixture of fatty acidglycerides or cocoa butter can be used.

In practicing the methods of the disclosure, therapeutically-effectiveamounts of the compounds described herein can be administered inpharmaceutical compositions to a subject having a disease or conditionto be treated. A therapeutically-effective amount can vary widelydepending on the severity of the disease, the age and relative health ofthe subject, the potency of the compounds used, and other factors. Thecompounds can be used singly or in combination with one or moretherapeutic agents as components of mixtures.

Pharmaceutical compositions can be formulated using one or morephysiologically-acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the molecule intopreparations that can be used pharmaceutically. Formulation can bemodified depending upon the route of administration chosen.Pharmaceutical compositions comprising a molecule described herein canbe manufactured, for example, by mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orcompression processes.

The pharmaceutical compositions can include at least onepharmaceutically acceptable carrier, diluent, or excipient and moleculedescribed herein as free-base or pharmaceutically-acceptable salt form.The methods and pharmaceutical compositions described herein include theuse crystalline forms (also known as polymorphs), and active metabolitesof these compounds having the same type of activity.

Methods for the preparation of compositions comprising the compoundsdescribed herein can include formulating the molecule with one or moreinert, pharmaceutically-acceptable excipients or carriers to form asolid, semi-solid, or liquid composition. Solid compositions caninclude, for example, powders, tablets, dispersible granules, capsules,cachets, and suppositories. Liquid compositions can include, forexample, solutions in which a compound is dissolved, emulsionscomprising a compound, or a solution containing liposomes, micelles, ornanoparticles comprising a compound as disclosed herein. Semi-solidcompositions can include, for example, gels, suspensions and creams. Thecompositions can be in liquid solutions or suspensions, solid formssuitable for solution or suspension in a liquid prior to use, or asemulsions. These compositions can also contain minor amounts ofnontoxic, auxiliary substances, such as wetting or emulsifying agents,pH buffering agents, and other pharmaceutically-acceptable additives.

Non-limiting examples of dosage forms can include feed, food, pellet,lozenge, liquid, elixir, aerosol, inhalant, spray, powder, tablet, pill,capsule, gel, geltab, nanosuspension, nanoparticle, microgel,suppository troches, aqueous or oily suspensions, ointment, patch,lotion, dentifrice, emulsion, creams, drops, dispersible powders orgranules, emulsion in hard or soft gel capsules, syrups, phytoceuticals,and nutraceuticals, or any combination thereof.

Non-limiting examples of pharmaceutically-acceptable excipients caninclude granulating agents, binding agents, lubricating agents,disintegrating agents, sweetening agents, glidants, anti-adherents,anti-static agents, surfactants, anti-oxidants, gums, coating agents,coloring agents, flavouring agents, coating agents, plasticizers,preservatives, suspending agents, emulsifying agents, plant cellulosicmaterial, and spheronization agents, or any combination thereof.

A composition can be, for example, an immediate release form or acontrolled release formulation. An immediate release formulation can beformulated to allow the molecules to act rapidly. Non-limiting examplesof immediate release formulations can include readily dissolvableformulations. A controlled release formulation can be a pharmaceuticalformulation that has been adapted such that drug release rates and drugrelease profiles can be matched to physiological and chronotherapeuticrequirements or, alternatively, has been formulated to effect release ofa drug at a programmed rate. Non-limiting examples of controlled releaseformulations can include granules, delayed release granules, hydrogels(e.g., of synthetic or natural origin), other gelling agents (e.g.,gel-forming dietary fibers), matrix-based formulations (e.g.,formulations comprising a polymeric material having at least one activeingredient dispersed through), granules within a matrix, polymericmixtures, granular masses, and the like.

A controlled release formulation can be a delayed release form. Adelayed release form can be formulated to delay a molecule's action foran extended period of time. A delayed release form can be formulated todelay the release of an effective dose of one or more molecules, forexample, for about 4, about 8, about 12, about 16, or about 24 hours.

A controlled release formulation can be a sustained release form. Asustained release form can be formulated to sustain, for example, themolecule's action over an extended period of time. A sustained releaseform can be formulated to provide an effective dose of any moleculedescribed herein (e.g., provide a physiologically-effective bloodprofile) over about 4, about 8, about 12, about 16 or about 24 hours.

Methods of Administration and Treatment Methods.

Pharmaceutical compositions containing molecules described herein can beadministered for prophylactic and/or therapeutic treatments. Intherapeutic applications, the compositions can be administered to asubject already suffering from a disease or condition, in an amountsufficient to cure or at least partially arrest the symptoms of thedisease or condition, or to cure, heal, improve, or ameliorate thecondition. Amounts effective for this use can vary based on the severityand course of the disease or condition, previous therapy, the subject'shealth status, weight, and response to the drugs, and the judgment ofthe treating physician.

Multiple therapeutic agents can be administered in any order orsimultaneously. If simultaneously, the multiple therapeutic agents canbe provided in a single, unified form, or in multiple forms, forexample, as multiple separate pills. The molecules can be packedtogether or separately, in a single package or in a plurality ofpackages. One or all of the therapeutic agents can be given in multipledoses. If not simultaneous, the timing between the multiple doses mayvary to as much as about a month.

Molecules described herein can be administered before, during, or afterthe occurrence of a disease or condition, and the timing ofadministering the composition containing a compound can vary. Forexample, the pharmaceutical compositions can be used as a prophylacticand can be administered continuously to subjects with a propensity toconditions or diseases in order to prevent the occurrence of the diseaseor condition. The molecules and pharmaceutical compositions can beadministered to a subject during or as soon as possible after the onsetof the symptoms. The administration of the molecules can be initiatedwithin the first 48 hours of the onset of the symptoms, within the first24 hours of the onset of the symptoms, within the first 6 hours of theonset of the symptoms, or within 3 hours of the onset of the symptoms.The initial administration can be via any route practical, such as byany route described herein using any formulation described herein. Amolecule can be administered as soon as is practicable after the onsetof a disease or condition is detected or suspected, and for a length oftime necessary for the treatment of the disease, such as, for example,from about 1 month to about 3 months. The length of treatment can varyfor each subject.

A molecule can be packaged into a biological compartment. A biologicalcompartment comprising the molecule can be administered to a subject.Biological compartments can include, but are not limited to, viruses(lentivirus, adenovirus), nanospheres, liposomes, quantum dots,nanoparticles, microparticles, nanocapsules, vesicles, polyethyleneglycol particles, hydrogels, and micelles.

For example, a biological compartment can comprise a liposome. Aliposome can be a self-assembling structure comprising one or more lipidbilayers, each of which can comprise two monolayers containingoppositely oriented amphipathic lipid molecules. Amphipathic lipids cancomprise a polar (hydrophilic) headgroup covalently linked to one or twoor more non-polar (hydrophobic) acyl or alkyl chains. Energeticallyunfavorable contacts between the hydrophobic acyl chains and asurrounding aqueous medium induce amphipathic lipid molecules to arrangethemselves such that polar headgroups can be oriented towards thebilayer's surface and acyl chains are oriented towards the interior ofthe bilayer, effectively shielding the acyl chains from contact with theaqueous environment.

Examples of preferred amphipathic compounds used in liposomes caninclude phosphoglycerides and sphingolipids, representative examples ofwhich include phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, phosphatidic acid,phoasphatidylglycerol, palmitoyloleoyl phosphatidylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylcholine, distearoylphosphatidylcholine(DSPC), dilinoleoylphosphatidylcholine and egg sphingomyelin, or anycombination thereof.

A biological compartment can comprise a nanoparticle. A nanoparticle cancomprise a diameter of from about 40 nanometers to about 1.5micrometers, from about 50 nanometers to about 1.2 micrometers, fromabout 60 nanometers to about 1 micrometer, from about 70 nanometers toabout 800 nanometers, from about 80 nanometers to about 600 nanometers,from about 90 nanometers to about 400 nanometers, from about 100nanometers to about 200 nanometers.

In some instances, as the size of the nanoparticle increases, therelease rate can be slowed or prolonged and as the size of thenanoparticle decreases, the release rate can be increased.

The amount of albumin in the nanoparticles can range from about 5% toabout 85% albumin (v/v), from about 10% to about 80%, from about 15% toabout 80%, from about 20% to about 70% albumin (v/v), from about 25% toabout 60%, from about 30% to about 50%, or from about 35% to about 40%.The pharmaceutical composition can comprise up to 30, 40, 50, 60, 70 or80% or more of the nanoparticle. In some instances, the nucleic acidmolecules of the disclosure can be bound to the surface of thenanoparticle.

A biological compartment can comprise a virus. The virus can be adelivery system for the pharmaceutical compositions of the disclosure.Exemplary viruses can include lentivirus, retrovirus, adenovirus, herpessimplex virus I or II, parvovirus, reticuloendotheliosis virus, andadeno-associated virus (AAV). Pharmaceutical compositions of thedisclosure can be delivered to a cell using a virus. The virus caninfect and transduce the cell in vivo, ex vivo, or in vitro. In ex vivoand in vitro delivery, the transduced cells can be administered to asubject in need of therapy.

Pharmaceutical compositions can be packaged into viral delivery systems.For example, the compositions can be packaged into virions by a HSV-1helper virus-free packaging system.

Viral delivery systems (e.g., viruses comprising the pharmaceuticalcompositions of the disclosure) can be administered by direct injection,stereotaxic injection, intracerebroventricularly, by minipump infusionsystems, by convection, catheters, intravenous, parenteral,intraperitoneal, and/or subcutaneous injection, to a cell, tissue, ororgan of a subject in need. In some instances, cells can be transducedin vitro or ex vivo with viral delivery systems. The transduced cellscan be administered to a subject having a disease. For example, a stemcell can be transduced with a viral delivery system comprising apharmaceutical composition and the stem cell can be implanted in thepatient to treat a disease. In some instances, the dose of transducedcells given to a subject can be about 1×10⁵ cells/kg, about 5×10⁵cells/kg, about 1×10⁶ cells/kg, about 2×10⁶ cells/kg, about 3×10⁶cells/kg, about 4×10⁶ cells/kg, about 5×10⁶ cells/kg, about 6×10⁶cells/kg, about 7×10⁶ cells/kg, about 8×10⁶ cells/kg, about 9×10⁶cells/kg, about 1×10⁷ cells/kg, about 5×10⁷ cells/kg, about 1×10⁸cells/kg, or more in one single dose.

Pharmaceutical compositions in biological compartments can be used totreatinflammatory diseases such as arthritis, cancers, such as, forexample, bone cancer, breast cancer, skin cancer, prostate cancer, livercancer, lung cancer, throat cancer and kidney cancer, bacterialinfections, to treat nerve damage, lung, liver and kidney diseases, eyetreatment, spinal cord injuries, heart disease, arterial disease.

Introduction of the biological compartments into cells can occur byviral or bacteriophage infection, transfection, conjugation, protoplastfusion, lipofection, electroporation, calcium phosphate precipitation,polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediatedtransfection, liposome-mediated transfection, particle gun technology,calcium phosphate precipitation, direct micro-injection,nanoparticle-mediated nucleic acid delivery, and the like.

Dosage

Pharmaceutical compositions described herein can be in unit dosage formssuitable for single administration of precise dosages. In unit dosageform, the formulation can be divided into unit doses containingappropriate quantities of one or more compounds. The unit dosage can bein the form of a package containing discrete quantities of theformulation. Non-limiting examples can include packaged tablets orcapsules, and powders in vials or ampoules. Aqueous suspensioncompositions can be packaged in single-dose non-reclosable containers.Multiple-dose reclosable containers can be used, for example, incombination with a preservative. Formulations for parenteral injectioncan be presented in unit dosage form, for example, in ampoules, or inmulti-dose containers with a preservative.

A molecule described herein can be present in a composition in a rangeof from about 1 mg to about 2000 mg; from about 5 mg to about 1000 mg,from about 10 mg to about 25 mg to 500 mg, from about 50 mg to about 250mg, from about 100 mg to about 200 mg, from about 1 mg to about 50 mg,from about 50 mg to about 100 mg, from about 100 mg to about 150 mg,from about 150 mg to about 200 mg, from about 200 mg to about 250 mg,from about 250 mg to about 300 mg, from about 300 mg to about 350 mg,from about 350 mg to about 400 mg, from about 400 mg to about 450 mg,from about 450 mg to about 500 mg, from about 500 mg to about 550 mg,from about 550 mg to about 600 mg, from about 600 mg to about 650 mg,from about 650 mg to about 700 mg, from about 700 mg to about 750 mg,from about 750 mg to about 800 mg, from about 800 mg to about 850 mg,from about 850 mg to about 900 mg, from about 900 mg to about 950 mg, orfrom about 950 mg to about 1000 mg.

A molecule described herein can be present in a composition in an amountof about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 10mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg,about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg,about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg,about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg,about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg,about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg,about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg,about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850mg, about 1900 mg, about 1950 mg, or about 2000 mg.

A molecule (e.g., site-directed polypeptide, nucleic acid-targetingnucleic acid and/or complex of a site-directed polypeptide and a nucleicacid-targeting nucleic acid) described herein can be present in acomposition that provides at least 0.1, 0.5, 1, 1.5, 2, 2.5 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 10 or more units of activity/mg molecule. In someembodiments, the total number of units of activity of the moleculedelivered to a subject is at least 25,000, 30,000, 35,000, 40,000,45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 110,000, 120,000,130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000,210,000, 220,000, 230,000, or 250,000 or more units. In someembodiments, the total number of units of activity of the moleculedelivered to a subject is at most 25,000, 30,000, 35,000, 40,000,45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 110,000, 120,000,130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000,210,000, 220,000, 230,000, or 250,000 or more units.

In some embodiments, at least about 10,000 units of activity isdelivered to a subject, normalized per 50 kg body weight. In someembodiments, at least about 10,000, 15,000, 25,000, 30,000, 35,000,40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 110,000,120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000,200,000, 210,000, 220,000, 230,000, or 250,000 units or more of activityof the molecule is delivered to the subject, normalized per 50 kg bodyweight. In some embodiments, a therapeutically effective dose comprisesat least 5×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4, 10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶,9×10⁶, 1×10⁷, 1.1×10⁷, 1.2×10⁷, 1.5×10⁷, 1.6×10⁷, 1.7×10⁷, 1.8×10⁷,1.9×10⁷, 2×10⁷, 2.1×10⁷, or 3×10⁷ or more units of activity of themolecule. In some embodiments, a therapeutically effective dosecomprises at most 5×10⁵ 1×10⁶, 2×10⁶, 3×10⁶, 4, 10⁶, 5×10⁶, 6×10⁶,7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 1.1×10⁷, 1.2×10⁷, 1.5×10⁷, 1.6×10⁷, 1.7×10⁷,1.8×10⁷, 1.9×10⁷, 2×10⁷, 2.1×10⁷, or 3×10⁷ or more units of activity ofthe molecule.

In some embodiments, a therapeutically effective dose is at least about10,000, 15,000, 20,000, 22,000, 24,000, 25,000, 30,000, 40,000, 50,000,60,000, 70,000, 80,000, 90,000, 100,000, 125,000, 150,000, 200,000, or500,000 units/kg body weight. In some embodiments, a therapeuticallyeffective dose is at most about 10,000, 15,000, 20,000, 22,000, 24,000,25,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000,125,000, 150,000, 200,000, or 500,000 units/kg body weight.

In some embodiments, the activity of the molecule delivered to a subjectis at least 10,000, 11,000, 12,000, 13,000, 14,000, 20,000, 21,000,22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 30,000, 32,000,34,000, 35,000, 36,000, 37,000, 40,000, 45,000, or 50,000 or more U/mgof molecule. In some embodiments, the activity of the molecule deliveredto a subject is at most 10,000, 11,000, 12,000, 13,000, 14,000, 20,000,21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 30,000,32,000, 34,000, 35,000, 36,000, 37,000, 40,000, 45,000, or 50,000 ormore U/mg of molecule.

Pharmacokinetic and Pharmacodynamic Measurements

Pharmacokinetic and pharmacodynamic data can be obtained by variousexperimental techniques. Appropriate pharmacokinetic and pharmacodynamicprofile components describing a particular composition can vary due tovariations in drug metabolism in human subjects. Pharmacokinetic andpharmacodynamic profiles can be based on the determination of the meanparameters of a group of subjects. The group of subjects includes anyreasonable number of subjects suitable for determining a representativemean, for example, 5 subjects, 10 subjects, 15 subjects, 20 subjects, 25subjects, 30 subjects, 35 subjects, or more. The mean can be determinedby calculating the average of all subject's measurements for eachparameter measured. A dose can be modulated to achieve a desiredpharmacokinetic or pharmacodynamics profile, such as a desired oreffective blood profile, as described herein.

The pharmacokinetic parameters can be any parameters suitable fordescribing a molecule. For example, the C_(max) can be, for example, notless than about 25 ng/mL; not less than about 50 ng/mL; not less thanabout 75 ng/mL; not less than about 100 ng/mL; not less than about 200ng/mL; not less than about 300 ng/mL; not less than about 400 ng/mL; notless than about 500 ng/mL; not less than about 600 ng/mL; not less thanabout 700 ng/mL; not less than about 800 ng/mL; not less than about 900ng/mL; not less than about 1000 ng/mL; not less than about 1250 ng/mL;not less than about 1500 ng/mL; not less than about 1750 ng/mL; not lessthan about 2000 ng/mL; or any other C_(max) appropriate for describing apharmacokinetic profile of a molecule described herein.

The T_(max) of a molecule described herein can be, for example, notgreater than about 0.5 hours, not greater than about 1 hours, notgreater than about 1.5 hours, not greater than about 2 hours, notgreater than about 2.5 hours, not greater than about 3 hours, notgreater than about 3.5 hours, not greater than about 4 hours, notgreater than about 4.5 hours, not greater than about 5 hours, or anyother T_(max) appropriate for describing a pharmacokinetic profile of amolecule described herein.

The AUC_((0-inf)) of a molecule described herein can be, for example,not less than about 50 ng·hr/mL, not less than about 100 ng/hr/mL, notless than about 150 ng/hr/mL, not less than about 200 ng·hr/mL, not lessthan about 250 ng/hr/mL, not less than about 300 ng/hr/mL, not less thanabout 350 ng/hr/mL, not less than about 400 ng/hr/mL, not less thanabout 450 ng/hr/mL, not less than about 500 ng/hr/mL, not less thanabout 600 ng/hr/mL, not less than about 700 ng/hr/mL, not less thanabout 800 ng/hr/mL, not less than about 900 ng/hr/mL, not less thanabout 1000 ng·hr/mL, not less than about 1250 ng/hr/mL, not less thanabout 1500 ng/hr/mL, not less than about 1750 ng/hr/mL, not less thanabout 2000 ng/hr/mL, not less than about 2500 ng/hr/mL, not less thanabout 3000 ng/hr/mL, not less than about 3500 ng/hr/mL, not less thanabout 4000 ng/hr/mL, not less than about 5000 ng/hr/mL, not less thanabout 6000 ng/hr/mL, not less than about 7000 ng/hr/mL, not less thanabout 8000 ng/hr/mL, not less than about 9000 ng/hr/mL, not less thanabout 10,000 ng/hr/mL, or any other AUC_((0-inf)) appropriate fordescribing a pharmacokinetic profile of a molecule described herein.

The plasma concentration of a molecule described herein about one hourafter administration can be, for example, not less than about 25 ng/mL,not less than about 50 ng/mL, not less than about 75 ng/mL, not lessthan about 100 ng/mL, not less than about 150 ng/mL, not less than about200 ng/mL, not less than about 300 ng/mL, not less than about 400 ng/mL,not less than about 500 ng/mL, not less than about 600 ng/mL, not lessthan about 700 ng/mL, not less than about 800 ng/mL, not less than about900 ng/mL, not less than about 1000 ng/mL, not less than about 1200ng/mL, or any other plasma concentration of a molecule described herein.

The pharmacodynamic parameters can be any parameters suitable fordescribing pharmaceutical compositions of the disclosure. For example,the pharmacodynamic profile can exhibit decreases in factors associatedwith inflammation after, for example, about 2 hours, about 4 hours,about 8 hours, about 12 hours, or about 24 hours.

Pharmaceutically-Acceptable Salts

The disclosure provides the use of pharmaceutically-acceptable salts ofany molecule described herein. Pharmaceutically-acceptable salts caninclude, for example, acid-addition salts and base-addition salts. Theacid that is added to the compound to form an acid-addition salt can bean organic acid or an inorganic acid. A base that is added to thecompound to form a base-addition salt can be an organic base or aninorganic base. In some embodiments, a pharmaceutically-acceptable saltis a metal salt. In some embodiments, a pharmaceutically-acceptable saltis an ammonium salt.

Metal salts can arise from the addition of an inorganic base to acompound of the invention. The inorganic base consists of a metal cationpaired with a basic counterion, such as, for example, hydroxide,carbonate, bicarbonate, or phosphate. The metal can be an alkali metal,alkaline earth metal, transition metal, or main group metal. In someembodiments, the metal is lithium, sodium, potassium, cesium, cerium,magnesium, manganese, iron, calcium, strontium, cobalt, titanium,aluminum, copper, cadmium, or zinc.

In some embodiments, a metal salt is a lithium salt, a sodium salt, apotassium salt, a cesium salt, a cerium salt, a magnesium salt, amanganese salt, an iron salt, a calcium salt, a strontium salt, a cobaltsalt, a titanium salt, an aluminum salt, a copper salt, a cadmium salt,or a zinc salt, or any combination thereof.

Ammonium salts can arise from the addition of ammonia or an organicamine to a compound of the invention. In some embodiments, the organicamine is triethyl amine, diisopropyl amine, ethanol amine, diethanolamine, triethanol amine, morpholine, N-methylmorpholine, piperidine,N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine,pyridine, pyrrazole, pipyrrazole, imidazole, pyrazine, or pipyrazine, orany combination thereof.

In some embodiments, an ammonium salt is a triethyl amine salt, adiisopropyl amine salt, an ethanol amine salt, a diethanol amine salt, atriethanol amine salt, a morpholine salt, an N-methylmorpholine salt, apiperidine salt, an N-methylpiperidine salt, an N-ethylpiperidine salt,a dibenzylamine salt, a piperazine salt, a pyridine salt, a pyrrazolesalt, a pipyrrazole salt, an imidazole salt, a pyrazine salt, or apipyrazine salt, or any combination thereof.

Acid addition salts can arise from the addition of an acid to a moleculeof the disclosure. In some embodiments, the acid is organic. In someembodiments, the acid is inorganic. In some embodiments, the acid ishydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid,nitrous acid, sulfuric acid, sulfurous acid, a phosphoric acid,isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbicacid, gentisinic acid, gluconic acid, glucaronic acid, saccaric acid,formic acid, benzoic acid, glutamic acid, pantothenic acid, acetic acid,propionic acid, butyric acid, fumaric acid, succinic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid, citric acid, oxalic acid, or maleic acid, or anycombination thereof.

In some embodiments, the salt is a hydrochloride salt, a hydrobromidesalt, a hydroiodide salt, a nitrate salt, a nitrite salt, a sulfatesalt, a sulfite salt, a phosphate salt, isonicotinate salt, a lactatesalt, a salicylate salt, a tartrate salt, an ascorbate salt, agentisinate salt, a gluconate salt, a glucaronate salt, a saccaratesalt, a formate salt, a benzoate salt, a glutamate salt, a pantothenatesalt, an acetate salt, a propionate salt, a butyrate salt, a fumaratesalt, a succinate salt, a methanesulfonate salt, an ethanesulfonatesalt, a benzenesulfonate salt, a p-toluenesulfonate salt, a citratesalt, an oxalate salt, or a maleate salt, or any combination thereof.

Engineered Site-Directed Polypeptides

General Overview

The disclosure describes methods, compositions, systems, and/or kits formodifying site-directed polypeptides (e.g., Cas9, Csy4, Cas5, Cas6,Argonaut, etc.) and/or related enzymes. Modifications may include anycovalent or non-covalent modification to site-directed polypeptides. Insome cases, this may include chemical modifications to one or moreregions of the site-directed polypeptide. In some cases, modificationsmay include conservative or non-conservative amino acid substitutions ofthe site-directed polypeptide. In some cases, modifications may includethe addition, deletion or substitution of any portion of thesite-directed polypeptide with amino acids, peptides, or domains thatare not found in the native site-directed polypeptide. In some cases,one or more non-native domains may be added, deleted or substituted inthe site-directed polypeptide. In some cases the site-directedpolypeptide may exist as a fusion protein.

In some cases, the present disclosure provides for the engineering ofsite-directed polypeptides to recognize a desired target nucleic acidsequence with desired enzyme specificity and/or activity. Modificationsto a site-directed polypeptide can be performed through proteinengineering. Protein engineering can include fusing functional domainsto such engineered site-directed polypeptide which can be used to modifythe functional state of the overall site-directed polypeptide or theactual target nucleic acid sequence of an endogenous cellular locus. Thesite-directed polypeptide of the disclosure can be used to regulateendogenous gene expression, both through activation and repression ofendogenous gene transcription.

The site-directed polypeptide-fusions can also be linked to otherregulatory or functional domains, for example nucleases, transposases ormethylases, to modify endogenous chromosomal sequences. In some cases,the site-directed polypeptide may be linked to at least one or moreregulatory domains, described herein. Non-limiting examples ofregulatory or functional domains include transcription factor repressoror activator domains such as KRAB and VP16, co-repressor andco-activator domains, DNA methyl transferases, histoneacetyltransferases, histone deacetylases, and DNA cleavage domains suchas the cleavage domain from the endonuclease FokI.

In some instances, one or more specific domains, regions or structuralelements of the site-directed polypeptide can be modified together.Modifications to the site-directed polypeptide may occur, but are notlimited to site-directed polypeptide elements such as regions thatrecognize or bind to the spacer-adjacent motif (PAM), and/or regionsthat bind or recognize the nucleic acid-targeting nucleic acid. Suchbinding or recognition elements may include a conserved bridging helix,highly basic region, N-terminal region, C-terminal region, RuvC motifs(e.g., RuvC and/or RuvC-like nuclease domains) and one or more nucleasedomains, such as HNH and/or HNH-like domains. Modifications may be madeto additional domains, structural elements, sequence or amino acidswithin the site-directed polypeptide.

Modifications to one or more region of the site-directed polypeptide maybe performed to alter various properties of the site-directedpolypeptide. In some cases, modifications may alter binding recognitionfor certain nucleic acid target sequences. This may include but is notlimited to increasing binding affinity and/or specificity to certainsequences or preferentially targeting of certain target nucleic acidsequences/recognition elements. In some cases, modifications may be usedto alter native nuclease function. In some cases, modifications to thesite-directed polypeptide may alter PAM specificity, tracrRNAspecificity, crRNA specificity, or specificity for additional nucleicacid elements, such as a nucleic acid-targeting nucleic acid.

Described herein are also compositions and methods including fusionproteins comprising a site-directed polypeptide (e.g., Cas9) and one ormore domains or regions engineered for genomic editing (e.g., cleavingof genes; alteration of genes, for example by cleavage followed byinsertion (physical insertion or insertion via homology-directed repair)of an exogenous sequence and/or cleavage followed by NHEJ; partial orcomplete inactivation of one or more genes; generation of alleles withaltered functional states of endogenous genes, insertion of regulatoryelements; etc.) and alterations of the genome which are carried into thegermline. Also disclosed are methods of making and using thesecompositions (i.e. reagents), for example to edit (i.e. alter) one ormore genes in a target cell. Thus, the methods and compositionsdescribed herein provide highly efficient methods for targeted genealteration (e.g., knock-in) and/or knockout (partial or complete) of oneor more genes and/or for randomized mutation of the sequence of anytarget allele, and, therefore, allow for the generation of animal modelsof human diseases. One skilled in the art will recognize that althoughthe term “genome engineering” or “genomic editing” is often used todescribe the methods herein, the methods and compositions describedherein can also be used to alter any target nucleic acid that may not bestrictly speaking in the genome of a cell (e.g. can be used on asynthetic nucleic acid, a plasmid, a vector, a viral nucleic acid, arecombinant nucleic acid, etc.).

The methods and compositions described herein allow for noveltherapeutic applications, (e.g., prevention and/or treatment of: geneticdiseases, cancer, fungal, protozoal, bacterial, and viral infection,ischemia, vascular disease, arthritis, immunological disorders, etc.),novel diagnostics (e.g. prediction and/or diagnosis of a condition) aswell as providing for research tools (e.g. kits, functional genomicsassays, and generating engineered cell lines and animal models forresearch and drug screening), and means for developing plants withaltered phenotypes, including but not limited to, increased diseaseresistance, and altering fruit ripening characteristics, sugar and oilcomposition, yield, and color. The methods and compositions describedherein allow for novel epigenetic studies.

Protein Modifications and Engineering

Amino Acid Alterations

Site-directed polypeptides, as disclosed herein, can be modified. Themodification can comprise modifications to an amino acid of thesite-directed polypeptide. The modifications can alter the primary aminoacid sequence and/or the secondary, tertiary, and quaternary amino acidstructure. In some cases some amino acid sequences of site-directedpolypeptide of the invention can be varied without a significant effecton the structure or function of the protein. The type of mutation may becompletely unimportant if the alteration occurs in some regions (e.g. anon-critical) region of the protein. In some cases, depending upon thelocation of the replacement, the mutation may not have a major effect onthe biological properties of the resulting variant. For example,properties and functions of the Cas9 variants can be of the same type aswild-type Cas9. In some cases, the mutation can critically impact thestructure and/or function of the site-directed polypeptide.

The location of where to modify a site-directed polypeptide (e.g., aCas9 variant) can be determined using sequence and/or structuralalignment. Sequence alignment can identify regions of a polypeptide thatsimilar and/or dissimilar (e.g., conserved, not conserved, hydrophobic,hydrophilic, etc). In some instances, a region in the sequence ofinterest that is similar to other sequences is suitable formodification. In some instances, a region in the sequence of interestthat is dissimilar from other sequences is suitable for modification.For example, sequence alignment can be performed by database search,pairwise alignment, multiple sequence alignment, genomic analysis, motiffinding, benchmarking, and/or programs such as BLAST, CS-BLAST, HHPRED,psi-BLAST, LALIGN, PyMOL, and SEQALN. Structural alignment can beperformed by programs such as Dali, PHYRE, Chimera, COOT, O, and PyMOL.Alignment can be performed by database search, pairwise alignment,multiple sequence alignment, genomic analysis, motif finding, or benchmarking, or any combination thereof.

A site-directed polypeptide can be modified to increase bindingspecificity to a nucleic acid-targeting nucleic acid and/or a targetnucleic acid. A site-directed polypeptide can be modified to increasebinding to specific regions of a nucleic acid-targeting nucleic acid(e.g., the spacer extension, the spacer, the minimum CRISPR repeat, theminimum tracrRNA sequence, the 3′ tracrRNA sequence, the tracrRNAextension) and/or a target nucleic acid.

In some cases, the modification can comprise a conservativemodification. A conservative amino acid change can involve substitutionof one of a family of amino acids which are related in their side chains(e.g, cysteine/serine)

In some cases amino acid changes in the Cas9 protein disclosed hereinare non-conservative amino acid changes, (i.e., substitutions ofdissimilar charged or uncharged amino acids). A non-conservative aminoacid change can involve substitution of one of a family of amino acidswhich may be unrelated in their side chains or a substitution thatalters biological activity of the site-directed polypeptide.

The mutation of amino acids can also change the selectivity of bindingto a target nucleic acid. The mutation may result in a change that maycomprise a change in the dissociation constant (Kd) of binding between amutated site-directed polypeptide and a target nucleic acid. The changein Kd of binding between a mutated site-directed polypeptide and atarget nucleic acid may be more than 1000-fold, more than 500-fold, morethan 100-fold, more than 50-fold, more than 25-fold, more than 10-fold,more than 5-fold, more than 4-fold, more than 3-fold, more than 2-foldhigher or lower than the Kd of binding of binding between a non-mutatedsite-directed polypeptide and a target nucleic acid. The change in Kd ofbinding between a mutated site-directed polypeptide and a target nucleicacid may be less than 1000-fold, less than 500-fold, less than 100-fold,less than 50-fold, less than 25-fold, less than 10-fold, less than5-fold, less than 4-fold, less than 3-fold, less than 2-fold higher orlower than the Kd of binding of binding between a non-mutatedsite-directed polypeptide and a target nucleic acid.

The mutation may result in a change that may comprise a change in K_(d)of binding between a mutated site-directed polypeptide and a PAM motif.The change in K_(d) of binding between a mutated site-directedpolypeptide and a PAM motif may be more than 1000-fold, more than500-fold, more than 100-fold, more than 50-fold, more than 25-fold, morethan 10-fold, more than 5-fold, more than 4-fold, more than 3-fold, morethan 2-fold higher or lower than the K_(d) of binding between anon-mutated site-directed polypeptide and a PAM motif. The change inK_(d) of binding between a mutated site-directed polypeptide and a PAMmotif may be less than 1000-fold, less than 500-fold, less than100-fold, less than 50-fold, less than 25-fold, less than 10-fold, lessthan 5-fold, less than 4-fold, less than 3-fold, less than 2-fold higheror lower than the K_(d) of binding of binding between a non-mutatedsite-directed polypeptide and a PAM motif.

The mutation may result in a change that may comprise a change in K_(d)of the binding between a mutated site-directed polypeptide and a nucleicacid-targeting nucleic acid. The change in K_(d) of binding between amutated site-directed polypeptide and a nucleic acid-targeting nucleicacid may be more than 1000-fold, more than 500-fold, more than 100-fold,more than 50-fold, more than 25-fold, more than 10-fold, more than5-fold, more than 4-fold, more than 3-fold, more than 2-fold higher orlower than the K_(d) of binding between a non-mutated site-directedpolypeptide and a nucleic acid-targeting nucleic acid. The change inK_(d) of binding between a mutated site-directed polypeptide and anucleic acid-targeting nucleic acid acid may be less than 1000-fold,less than 500-fold, less than 100-fold, less than 50-fold, less than25-fold, less than 10-fold, less than 5-fold, less than 4-fold, lessthan 3-fold, less than 2-fold higher or lower than the K_(d) of bindingbetween a non-mutated site-directed polypeptide and a nucleicacid-targeting nucleic acid.

The mutation of a site-directed polypeptide can also change the kineticsof the enzymatic action of the site-directed polypeptide. The mutationmay result in a change that may comprise a change in the K_(m) of themutated site-directed polypeptide. The change in K_(m) of the mutatedsite-directed polypeptide may be more than 1000-fold, more than500-fold, more than 100-fold, more than 50-fold, more than 25-fold, morethan 10-fold, more than 5-fold, more than 4-fold, more than 3-fold, morethan 2-fold higher or lower than the K_(m) of a non-mutatedsite-directed polypeptide. The change in K_(m) of a mutatedsite-directed polypeptide may be less than 1000-fold, less than500-fold, less than 100-fold, less than 50-fold, less than 25-fold, lessthan 10-fold, less than 5-fold, less than 4-fold, less than 3-fold, lessthan 2-fold higher or lower than the K_(m) of a non-mutatedsite-directed polypeptide.

The mutation of a site-directed polypeptide may result in a change thatmay comprise a change in the turnover of the site-directed polypeptide.The change in the turnover of the mutated site-directed polypeptide maybe more than 1000-fold, more than 500-fold, more than 100-fold, morethan 50-fold, more than 25-fold, more than 10-fold, more than 5-fold,more than 4-fold, more than 3-fold, more than 2-fold higher or lowerthan the turnover of a non-mutated site-directed polypeptide. The changein the turnover of a mutated site-directed polypeptide may be less than1000-fold, less than 500-fold, less than 100-fold, less than 50-fold,less than 25-fold, less than 10-fold, less than 5-fold, less than4-fold, less than 3-fold, less than 2-fold higher or lower than theturnover of a non-mutated site-directed polypeptide.

The mutation may result in a change that may comprise a change in the ΔGof the enzymatic action of the site-directed polypeptide. The change inthe ΔG of the mutated site-directed polypeptide may be more than1000-fold, more than 500-fold, more than 100-fold, more than 50-fold,more than 25-fold, more than 10-fold, more than 5-fold, more than4-fold, more than 3-fold, more than 2-fold higher or lower than the ΔGof a non-mutated site-directed polypeptide. The change in the turnoverof a mutated site-directed polypeptide may be less than 1000-fold, lessthan 500-fold, less than 100-fold, less than 50-fold, less than 25-fold,less than 10-fold, less than 5-fold, less than 4-fold, less than 3-fold,less than 2-fold higher or lower than the ΔG of a non-mutatedsite-directed polypeptide.

The mutation may result in a change that may comprise a change in theV_(max) of the enzymatic action of the site-directed polypeptide. Thechange in the V_(max) of the mutated site-directed polypeptide may bemore than 1000-fold, more than 500-fold, more than 100-fold, more than50-fold, more than 25-fold, more than 10-fold, more than 5-fold, morethan 4-fold, more than 3-fold, more than 2-fold higher or lower than theV_(max) of a non-mutated site-directed polypeptide. The change in theturnover of a mutated site-directed polypeptide may be less than1000-fold, less than 500-fold, less than 100-fold, less than 50-fold,less than 25-fold, less than 10-fold, less than 5-fold, less than4-fold, less than 3-fold, less than 2-fold higher or lower than theV_(max) of a non-mutated site-directed polypeptide.

The mutation may result in a change that may comprise a change in anykinetic parameter of the site-directed polypeptide. The mutation mayresult in in a change that may comprise a change in any thermodynamicparameter of the site-directed polypeptide. The mutation may result inin a change that may comprise a change in the surface charge, surfacearea buried, and/or folding kinetics of the site-directed polypeptideand/or enzymatic action of the site-directed polypeptide.

Amino acids in the site-directed polypeptide of the present inventionthat are essential for function can be identified by methods such assite-directed mutagenesis, alanine-scanning mutagenesis, proteinstructure analysis, nuclear magnetic resonance, photoaffinity labeling,and electron tomography, high-throughput screening, ELISAs, biochemicalassays, binding assays, cleavage assays (e.g., Surveyor assay), reporterassays, and the like.

Other amino acid alterations may also include amino acids withglycosylated forms, aggregative conjugates with other molecules, andcovalent conjugates with unrelated chemical moieties (e.g., pegylatedmolecules). Covalent variants can be prepared by linking functionalitiesto groups which are found in the amino acid chain or at the N- orC-terminal residue. In some cases mutated site-directed polypeptides mayalso include allelic variants and species variants.

Truncations of regions which do not affect functional activity of theCas9 proteins may be engineered. Truncations of regions which do affectfunctional activity of the Cas9 protein may be engineered. A truncationmay comprise a truncation of less than 5, less than 10, less than 15,less than 20, less than 25, less than 30, less than 35, less than 40,less than 45, less than 50, less than 60, less than 70, less than 80,less than 90, less than 100 or more amino acids. A truncation maycomprise a truncation of more than 5, more than 10, more than 15, morethan 20, more than 25, more than 30, more than 35, more than 40, morethan 45, more than 50, more than 60, more than 70, more than 80, morethan 90, more than 100 or more amino acids. A truncation may comprisetruncation of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the site-directedpolypeptide.

Deletions of regions which do not affect functional activity of the Cas9proteins may be engineered. Deletions of regions which do affectfunctional activity of the Cas9 protein may be engineered. A deletioncan comprise a deletion of less than 5, less than 10, less than 15, lessthan 20, less than 25, less than 30, less than 35, less than 40, lessthan 45, less than 50, less than 60, less than 70, less than 80, lessthan 90, less than 100 or more amino acids. A deletion may comprise adeletion of more than 5, more than 10, more than 15, more than 20, morethan 25, more than 30, more than 35, more than 40, more than 45, morethan 50, more than 60, more than 70, more than 80, more than 90, morethan 100 or more amino acids. A deletion may comprise deletion of about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or 100% of the site-directed polypeptide. Adeletion can occur at the N-terminus, the C-terminus, or at any regionin the polypeptide chain.

Screens

The disclosure provides for methods for engineering a site-directedpolypeptide. Screens can be used to engineering a site-directedpolypeptide. For example, a screen can be set up to screen for theeffect of mutations in a region of the site-directed polypeptide. Forexample, a screen can be set up to test modifications of the highlybasic patch on the affinity for RNA structure (e.g., nucleicacid-targeting nucleic acid structure), or processing capability (e.g.,target nucleic acid cleavage). Exemplary screening methods can includebut are not limited to, cell sorting methods, mRNA display, phagedisplay, and directed evolution.

Fusions

In some instances, the site-directed polypeptide is modified such thatit comprises a non-native sequence (i.e. the polypeptide has amodification that alters it from the allele or sequence it was derivedfrom) (e.g., the polypeptide can be referred to as a fusion). Thenon-native sequence can also include one or more additional proteins,protein domains, subdomains or polypeptides. For example, Cas9 may befused with any suitable additional nonnative nucleic acid bindingproteins and/or domains, including but not limited to transcriptionfactor domains, nuclease domains, nucleic acid polymerizing domains. Thenon-native sequence can comprise a sequence of Cas9 and/or a Cas9homologue.

The non-native sequence can confer new functions to the fusion protein.These functions can include for example, DNA cleavage, DNA methylation,DNA damage, DNA repair, modification of a target polypeptide associatedwith target DNA (e.g., a histone, a DNA-binding protein, etc.), leadingto, for example, histone methylation, histone acetylation, histoneubiquitination, and the like. Other functions conferred by a fusionprotein can include methyltransferase activity, demethylase activity,deamination activity, dismutase activity, alkylation activity,depurination activity, oxidation activity, pyrimidine dimer formingactivity, integrase activity, transposase activity, recombinaseactivity, polymerase activity, ligase activity, helicase activity,photolyase activity or glycosylase activity, acetyltransferase activity,deacetylase activity, kinase activity, phosphatase activity, ubiquitinligase activity, deubiquitinating activity, adenylation activity,deadenylation activity, SUMOylating activity, deSUMOylating activity,ribosylation activity, deribosylation activity, myristoylation activity,remodelling activity, protease activity, oxidoreductase activity,transferase activity, hydrolase activity, lyase activity, isomeraseactivity, synthase activity, synthetase activity, and demyristoylationactivity, or any combination thereof.

Modifications to the Bridge Helix

In some cases, the bridge helix region of Cas9 may be modified (e.g., toalter PAM specificity). In some cases, the bridge helix may sharehomology with the bridge helix identified in the Cas9 protein of S.pyogenes (residues 551-566). In some cases, the bridge helix may shareat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% homology with residues551-556 of S. pyogenes Cas9 bridge helix. In some cases, the bridgehelix may share at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologywith residues 551-556 of S. pyogenes Cas9 bridge helix.

In some cases, modifications to the bridge helix may include but are notlimited to individual amino acid modifications, as described herein. Insome cases, modification to the bridge helix may include but are notlimited to insertions, deletions or substitution of individual aminoacids, or polypeptides, such as other protein elements (e.g domains,structural motifs, proteins).

Modifications may include modifications to at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids ofthe bridge helix. Modifications may include modifications to at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore amino acids of the bridge helix. Modifications may also include atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the bridge helix.Modifications may also include at most 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or100% of the bridge helix.

In some cases, modifications to site-directed polypeptide bridge helixsequences may include particular polypeptide structural motifs,including but not limited to alpha helix, beta strand, beta sheet,310-helix, pi-helix, polyproline I motif, polyproline II motif,polyproline III motif, beta turn, alpha-turn-alpha, or helix kinks orhinges. For example, substitutions to the site-directed polypeptidebridge helix may include substitution or addition with one or moreproline amino acid residues. Insertion of proline residues may introducekinks in the bridge helix which may alter the binding specificity of thebridge helix for the PAM. In another example, substitution or additionmay include one or more glycine amino acid residues. Insertion orsubstitution of glycine residues may introduce increased flexibility inthe bridge helix, or “hinges” which may also alter the bindingspecificity of the bridge helix for the PAM. Altering bindingspecificity may or may not affect enzymatic activity of the Cas9protein.

In some cases, modifications to site-directed polypeptide bridge helixsequences may include deletion of at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99%, or 100% of the bridge helix. In some cases, modifications tosite-directed polypeptide bridge helix sequences may include deletion ofat most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the bridgehelix.

In some cases, modifications to site-directed polypeptide bridge helixsequences may include addition or substitution of at least 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% of a homologous site-directedpolypeptide bridge helix. In some cases, modifications to site-directedpolypeptide bridge helix sequences may include addition or substitutionof at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of ahomologous site-directed polypeptide bridge helix.

For example, nonnative Cas9 bridge helices may be derived from anysuitable organism. In some cases, the Cas9 protein and bridge helix maybe derived from prokaryotic organisms, including but not limited toarchea, bacteria, protists (e.g., E. coli, S. pyogenes, S. thermophilus,P. furiosus, and etc.).

For example, the bridge helix of the S. pyogenes Cas9 enzyme may besubstituted or inserted with the bridge helix, or fragment thereof,derived from another Cas9 enzyme from a different species.

In some instances, a site-directed polypeptide comprises an amino acidsequence comprising at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid-cleaving domains (i.e., a HNH domain and RuvCdomain), and a modified bridge helix.

Modifications to the Highly Basic Patch

PAM binding and specificity may also be affected by additional regionswithin the Cas9 protein. In some cases, a highly basic patch or region,comprising basic amino acid residues adjacent to the PAM binding site,may also be modified to alter PAM specificity. In some cases, the highlybasic patch or region may share homology with the highly basic patchidentified in the Cas9 of S. pyogenes contained within N-terminalregion, or amino acid residues 1-270 of the S. pyogenes Cas9. In somecases, the highly basic patch may share at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99%, or 100% homology with the S. pyogenes Cas9 highly basic patch.In some cases, the highly basic patch may share at most 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, 99%, or 100% homology with the S. pyogenes Cas9 highlybasic patch.

In some cases, modifications to the highly basic patch may include butare not limited to individual amino acid modifications, as describedherein. In some cases, modification to the highly basic patch mayinclude but are not limited to insertions, deletions or substitution ofindividual amino acids, or polypeptides, such as other protein elements(e.g domains, structural motifs, proteins).

Modifications may include modifications to at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 21, 42, 43,44, 45, 46, 47, 48, 49, 50 or more amino acids of the highly basicpatch. Modifications may include modifications to at most 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 21, 42,43, 44, 45, 46, 47, 48, 49, 50 or more amino acids of the highly basicpatch. Modifications may also include at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99%, or 100% of the highly basic patch. Modifications may alsoinclude at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the highlybasic patch.

In some cases, modifications to highly basic patch sequence of thesite-directed polypeptide may include particular polypeptide structuralmotifs, including but not limited to alpha helix, beta strand, betasheet, 310-helix, pi-helix, polyproline I motif, polyproline II motif,polyproline III motif, beta turn, alpha-turn-alpha, or helix kinks orhinges.

Substitutions to the highly basic patch of the site-directed polypeptidemay include substitution or addition with one or more acidic amino acidresidues. Insertion of acidic residues may decrease the overall basiccharge of this area of the site-directed polypeptide and may alter thebinding specificity of the highly basic patch for the PAM. In anotherexample, substitution or addition may include one or more basic aminoacid residues. Insertion or substitution of basic residues may increasethe charge area or ionic strength of intereraction between thepolypeptide and the nucleic acid and may also alter the bindingspecificity of the highly basic patch for the PAM. Altering bindingspecificity may or may not affect enzymatic activity of thesite-directed polypeptide.

In some cases, modifications to the site-directed polypeptide highlybasic patch sequences may include deletion of at least 1%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, 99%, or 100% of the highly basic patch. In some cases,modifications to site-directed polypeptide highly basic patch sequencesmay include deletion of at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or100% of the highly basic patch.

In some cases, modifications to site-directed polypeptide highly basicpatch sequences may include addition or substitution of at least 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98%, 99%, or 100% of a homologous Cas9 highly basicpatch. In some cases, modifications to the site-directed polypeptidehighly basic patch sequences may include addition or substitution of atmost 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of a homologous Cas9highly basic patch.

Homologous Cas9 highly basic patch sequences may be derived from anysuitable organism. In some cases, the Cas9 protein may be derived fromprokaryotic organisms such as archea, bacteria, protists (e.g., E. coli,S. pyogenes, S. thermophilus, P. furiosus, and etc.). For example, thehighly basic patch of the S. pyogenes Cas9 enzyme may be substituted orinserted with the highly basic patch, or fragment thereof, derived froma Cas9 of another species.

In some instances, a site-directed polypeptide comprises an amino acidsequence comprising at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid-cleaving domains (i.e., a HNH domain and RuvCdomain), and a modified highly basic patch.

Modifications to the HNH Domain

In some cases, the HNH domain in a site-directed polypeptide may bemodified to alter PAM specificity. In some cases, the HNH domain mayshare homology with the HNH domain identified in the C terminal domainof the Cas9 protein of S. pyogenes (residues 860-1100). In some cases,the HNH domain may share at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%homology with residues 551-556 of S. pyogenes Cas9 HNH domain. In somecases, the HNH domain may share at most 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,99%, or 100% homology with residues 860-1100 of S. pyogenes Cas9 HNHdomain.

In some cases, modifications to the HNH domain may include but are notlimited to individual amino acid modifications, as described herein. Insome cases, modification to the HNH domain may include but are notlimited to insertions, deletions or substitution of individual aminoacids, or polypeptides, such as other protein elements (e.g domains,structural motifs, proteins).

Modifications may include modifications to at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids ofthe HNH domain. Modifications may include modifications to at most 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moreamino acids of the HNH domain. Modifications may also include at least5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the HNH domain.Modifications may also include at most 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or100% of the HNH domain.

In some cases, modifications to site-directed polypeptide HNH domainsequences may include particular polypeptide structural motifs,including but not limited to alpha helix, beta strand, beta sheet,310-helix, pi-helix, polyproline I motif, polyproline II motif,polyproline III motif, beta turn, alpha-turn-alpha, or helix kinks orhinges.

Substitutions to the HNH domain of the site-directed polypeptide mayinclude substitution or addition with one or more amino acid residues.In some cases, the HNH domain may be replaced or fused with othersuitable nucleic acid binding domains. A nucleic acid-binding domain cancomprise RNA. There can be a single nucleic acid-binding domain.Examples of nucleic acid-binding domains can include, but are notlimited to, a helix-turn-helix domain, a zinc finger domain, a leucinezipper (bZIP) domain, a winged helix domain, a winged helix turn helixdomain, a helix-loop-helix domain, a HMG-box domain, a Wor3 domain, animmunoglobulin domain, a B3 domain, a TALE domain, a Zinc-finger domain,a RNA-recognition motif domain, a double-stranded RNA-binding motifdomain, a double-stranded nucleic acid binding domain, a single-strandednucleic acid binding domains, a KH domain, a PUF domain, a RGG boxdomain, a DEAD/DEAH box domain, a PAZ domain, a Piwi domain, and acold-shock domain, a RNAseH domain, a HNH domain, a RuvC-like domain, aRAMP domain, a Cas5 domain, a Cas6 domain.

In some cases, modifications to site-directed polypeptide HNH domainsequences may include deletion of at least 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99%, or 100% of the HNH domain. In some cases, modifications tosite-directed polypeptide HNH domain sequences may include deletion ofat most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the HNH domain.

In some cases, modifications to site-directed polypeptide HNH domainsequences may include addition or substitution of at least 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% of a homologous Cas9 HNH domain. Insome cases, modifications to site-directed polypeptide HNH domainsequences may include addition or substitution of at most 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% of a homologous Cas9 HNH domain.

Homologous Cas9 HNH domains may be derived from any suitable organism.In some cases, the Cas9 protein may be derived from prokaryoticorganisms such as archea, bacteria, protists (e.g., E. coli, S.pyogenes, S. thermophilus, P. furiosus, and etc.). For example, the HNHdomain of the S. pyogenes Cas9 enzyme may be substituted or insertedwith the HNH domain, or fragment thereof, derived from a Cas9 enzyme ofanother species. In some cases, at least one homologous Cas9 HNH domainmay be inserted into the HNH domain. In some cases, the at least onehomologous Cas9 HNH domain may form an HNH domain array, comprising atleast two HNH domains. In some cases, an HNH domain array can compriseat least one Cas9 HNH domain and at least one second HNH domain.

In some instances, the modification to the HNH or HNH-like domain cancomprise insertion of the same or similar HNH or HNH-like domain intandem (e.g., adjacent) to the HNH domain of Cas9. The HNH or HNH-likedomain can be inserted N-terminal and/or C-terminal of the HNH domain inCas9. Insertion of one or more HNH or HNH-like domains in Cas9 can beuseful in extending specificity in a target nucleic acid. Insertion ofone or more HNH or HNH-like domains in Cas9 can be useful in duplicatingspecificity in a target nucleic acid. For example, insertion of one ormore HNH or HNH-like domains can configure Cas9 to recognize a longerstretch of target nucleic acid, recognize a different RNA-DNA hybrid,and/or recognize a target nucleic acid with higher binding affinity.

In some instances, a site-directed polypeptide comprises an amino acidsequence comprising at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid-cleaving domains (i.e., a HNH domain and RuvCdomain), and a modified HNH domain.

Modifications to the RuvC or RuvC-Like Domains

In some cases, the RuvC or RuvC-like domain in the site-directedpolypeptide may be modified to alter PAM specificity. In some cases, theRuvC or RuvC-like domain may share homology with the RuvC or RuvC-likedomain identified in the Cas9 protein of S. pyogenes (residues 1-270).In some cases, the RuvC or RuvC-like domain may share at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% homology with residues 551-556 of S.pyogenes Cas9 RuvC or RuvC-like domain. In some cases, the RuvC orRuvC-like domain may share at most 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or100% homology with residues 1-270 of S. pyogenes Cas9 RuvC or RuvC-likedomain.

In some cases, modifications to the RuvC or RuvC-like domain may includebut are not limited to individual amino acid modifications, as describedherein. In some cases, modification to the RuvC or RuvC-like domain mayinclude but are not limited to insertions, deletions or substitution ofindividual amino acids, or polypeptides, such as other protein elements(e.g domains, structural motifs, proteins).

Modifications may include modifications to at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids ofthe RuvC or RuvC-like domain. Modifications may include modifications toat most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 or more amino acids of the RuvC or RuvC-like domain.Modifications may also include at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,99%, or 100% of the RuvC or RuvC-like domain. Modifications may alsoinclude at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the RuvC orRuvC-like domain.

In some cases, modifications to site-directed polypeptide RuvC orRuvC-like domain sequences may include particular polypeptide structuralmotifs, including but not limited to alpha helix, beta strand, betasheet, 310-helix, pi-helix, polyproline I motif, polyproline II motif,polyproline III motif, beta turn, alpha-turn-alpha, or helix kinks orhinges.

Substitutions to the site-directed polypeptide RuvC or RuvC-like domainmay include substitution or addition with one or more amino acidresidues. In some cases, the RuvC or RuvC-like domain may be replaced orfused with other suitable nucleic acid binding domains. A nucleicacid-binding domain can comprise RNA. There can be a single nucleicacid-binding domain. Examples of nucleic acid-binding domains caninclude, but are not limited to, a helix-turn-helix domain, a zincfinger domain, a leucine zipper (bZIP) domain, a winged helix domain, awinged helix turn helix domain, a helix-loop-helix domain, a HMG-boxdomain, a Wor3 domain, an immunoglobulin domain, a B3 domain, a TALEdomain, a Zinc-finger domain, a RNA-recognition motif domain, adouble-stranded RNA-binding motif domain, a double-stranded nucleic acidbinding domain, a single-stranded nucleic acid binding domains, a KHdomain, a PUF domain, a RGG box domain, a DEAD/DEAH box domain, a PAZdomain, a Piwi domain, a cold-shock domain, a RNAseH domain, a HNHdomain, a RuvC-like domain, a RAMP domain, a Cas5 domain, and a Cas6domain.

In some cases, modifications to site-directed polypeptide RuvC orRuvC-like domain sequences may include deletion of at least 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% of the RuvC or RuvC-like domain. Insome cases, modifications to site-directed polypeptide RuvC or RuvC-likedomain sequences may include deletion of at most 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, 99%, or 100% of the RuvC or RuvC-like domain.

In some cases, modifications to site-directed polypeptide RuvC orRuvC-like domain sequences may include addition or substitution of atleast 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of a homologousCas9 RuvC or RuvC-like domain. In some cases, modifications to thesite-directed polypeptide RuvC or RuvC-like domain sequences may includeaddition or substitution of at most 1%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,99%, or 100% of a homologous Cas9 RuvC or RuvC-like domain.

Homologus Cas9 RuvC or RuvC-like domains may be derived from anysuitable organism. In some cases, the Cas9 protein may be derived fromprokaryotic organisms such as archea, bacteria, protists (e.g., E. coli,S. pyogenes, S. thermophilus, P. furiosus and etc.). For example, theRuvC or RuvC-like domain of the S. pyogenes Cas9 enzyme may besubstituted or inserted with the RuvC or RuvC-like domain, or fragmentthereof, derived from another Cas9 enzyme, such as one from anotherspecies.

In some instances, the modification to the RuvC or RuvC-like domain cancomprise insertion of the same or similar RuvC or RuvC-like domain intandem (e.g., adjacent) to the RuvC or RuvC-like domain of Cas9. TheRuvC or RuvC-like domain can be inserted N-terminal and/or C-terminal ofthe RuvC or RuvC-like domain in Cas9. Insertion of one or more RuvC orRuvC-like domains in Cas9 can be useful in extending specificity in atarget nucleic acid. Insertion of one or more RuvC or RuvC-like domainsin Cas9 can be useful in duplicating specificity in a target nucleicacid. For example, insertion of one or more RuvC or RuvC-like domainscan configure Cas9 to recognize a longer stretch of target nucleic acid,recognize a different RNA-DNA hybrid, and/or recognize a target nucleicacid with higher binding affinity.

In some instances, a site-directed polypeptide comprises an amino acidsequence comprising at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid-cleaving domains (i.e., a HNH domain and RuvCdomain), and a modified RuvC domain.

Modifications to Cas9 Domains Containing RNA Polymerase HomologousRegions

In some cases, a site-directed polypeptide may be share homology withRNA polymerase. Both proteins may share similar functionally homologousdomains that are involved in catalysis of binding and manipulation ofnucleic acids. For example, RNA polymerase can comprise regions ofpolypeptide sequence that is involved in binding RNA-DNA duplexes. Insome cases, these regions may aid in melting the duplex.

In some cases, a site-directed polypeptide may also comprise certainregions that affect the binding specificity of the enzyme for nucleicacids. In some cases, these regions may share either sequence orfunctional homology with domains or regions as found in RNA polymerase.In some cases, the basic region of the N-terminus of the site-directedpolypeptide may be bind to the tracrRNA and crRNA or a single RNA(sgRNA). In S. pyogenes, this may correspond to a region of residues50-100.

Generally, the present disclosure provides for any suitable modificationto this region or adjacent regions. In some cases, the tracrRNA/crRNAbinding region (e.g., the nucleic acid-targeting nucleic acid bindingregion) in the site-directed polypeptide may be modified to alterspecificity for the nucleic acid. In some cases, the tracrRNA/crRNAbinding region may share homology with the tracrRNA/crRNA binding regionidentified in the Cas9 protein of S. pyogenes (residues 50-100). In somecases, the tracrRNA/crRNA binding region may share at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% homology with residues 5-100 of S.pyogenes Cas9 tracrRNA/crRNA binding region. In some cases, thetracrRNA/crRNA binding region may share at most 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99%, or 100% homology with residues 50-100 of S. pyogenes Cas9tracrRNA/crRNA binding region.

In some cases, modifications to the tracrRNA/crRNA binding region mayinclude but are not limited to individual amino acid modifications, asdescribed herein. In some cases, modification to the tracrRNA/crRNAbinding region may include but are not limited to insertions, deletionsor substitution of individual amino acids, or polypeptides, such asother protein elements (e.g domains, structural motifs, proteins).

Modifications may include modifications to at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids ofthe tracrRNA/crRNA binding region. Modifications may includemodifications to at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more amino acids of the tracrRNA/crRNA bindingregion. Modifications may also include at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99%, or 100% of the tracrRNA/crRNA binding region. Modificationsmay also include at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% ofthe tracrRNA/crRNA binding region.

In some cases, modifications to the site-directed polypeptidetracrRNA/crRNA binding region sequences may include particularpolypeptide structural motifs, including but not limited to alpha helix,beta strand, beta sheet, 310-helix, pi-helix, polyproline I motif,polyproline II motif, polyproline III motif, beta turn,alpha-turn-alpha, or helix kinks or hinges.

For example, substitutions to the site-directed polypeptidetracrRNA/crRNA binding region may include substitution or addition withone or more proteins or fragments thereof. For example, thetracrRNA/crRNA binding region could be substituted with the RNA-bindingdomain from any of the known RNA-binding Type I, Type II, or Type IIICRISPR system member. The tracrRNA/crRNA binding region could besubstituted with the RNA-binding domain from any of the knownRNA-binding member of the RAMP superfamily. The tracrRNA/crRNA bindingregion could be substituted with the RNA-binding domain from any of theknown RNA-binding member of the Cas7, Cas6, Cas5 families. In oneexample, the tracr RNA requirement may be replaced with the requirementfor a 5′ hairpin sequence with the spacer sequence placed downstream ofthe hairpin for DNA recognition.

In some cases, modifications to site-directed polypeptide tracrRNA/crRNAbinding region sequences may include deletion of at least 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% of the tracrRNA/crRNA binding region.In some cases, modifications to site-directed polypeptide tracrRNA/crRNAbinding region sequences may include deletion of at most 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% of the tracrRNA/crRNA binding region.

In some cases, modifications to site-directed polypeptide tracrRNA/crRNAbinding region sequences may include addition or substitution of atleast 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of a homologousCas9 tracrRNA/crRNA binding region. In some cases, modifications tosite-directed polypeptide tracrRNA/crRNA binding region sequences mayinclude addition or substitution of at most 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99%, or 100% of a homologous Cas9 tracrRNA/crRNA binding region.

Homologous site-directed polypeptide tracrRNA/crRNA binding regions maybe derived from any suitable organism. In some cases, the tracrRNA/crRNAbinding region may be derived from prokaryotic organisms, including butnot limited to archea, bacteria, protists (e.g., E. coli, S. pyogenes,S. thermophilus, P. furiosus and etc.). For example, the tracrRNA/crRNAbinding region of the S. pyogenes Cas9 may be substituted or insertedwith the tracrRNA/crRNA binding region, or fragment thereof, derivedfrom another Cas9, such as one derived from another species.

In some instances, a site-directed polypeptide comprises an amino acidsequence comprising at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid-cleaving domains (i.e., a HNH domain and RuvCdomain), and a modified polymerase-like domain.

Modifications to Alter PAM Specificity

In some instances, a site-directed polypeptide can recognize aprotospacer adjacent motif (PAM). A PAM can be any sequence in a targetnucleic acid that is recognized by a site-directed polypeptide and isimmediately 3′ of the target nucleic acid sequence targeted by thespacer of a nucleic acid-targeting nucleic acid. For example, a PAM cancomprise 5′-NGG-3′ or 5′-NGGNG-3′, 5′-NNAAAAW-3′, 5′-NNNNGATT-3′,5′-GNNNCNNA-3′, 5′-NNNACA-3′ where N is any nucleotide and N isimmediately 3′ of the target nucleic acid sequence targeted by thespacer sequence.

A site-directed polypeptide can be modified to alter PAM specificity.For example, a site-directed polypeptide can be modified such that priorto the modifying the polypeptide targets a first protospacer adjacentmotif and after the modifying the site-directed polypeptide targets asecond protospacer adjacent motif. In some instances, altered PAMspecificity can comprise a change in binding specificity (e.g.,increased binding, decreased binding), and/or a change in the bindingconstant (e.g., increase Kd, decrease Kd).

A site-directed polypeptide can be modified such that the site-directedpolypeptide can recognize a new type of PAM different from the the typethe wild-type site-directed polypeptide recognizes. For example, asite-directed polypeptide that recognizes the 5′-NGG-3′ PAM can bemodified such that it can recognize the 5′-NGGNG-3′ PAM, 5′-NNAAAAW-3′,5′-NNNNGATT-3′, 5′-GNNNCNNA-3′, or 5′-NNNACA-3′.

Any region of a site-directed polypeptide can be engineered (e.g.,bridge helix, HNH and/or HNH-like domain, RuvC and/or RuvC-like domain,basic patch) to alter PAM specificity according to the methods of thedisclosure.

Regions corresponding to residues 445-507, 446-497, 1096-1225, 1105-1138of a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes,SEQ ID NO: 8) can be engineered to modify PAM recognition. Engineeringof these regions can comprise introducing mutations, replacing withcorresponding regions from other Cas9 orthologues, deletions, insertionsetc. Regions corresponding to residues 718-757, 22-49, 65-95, 445-507,446-497, 1096-1225, 1105-1138 can be engineered to modify recognition ofthe nucleic-acid targeting nucleic acid. Regions corresponding toresidues 445-507 and 1105-1138 can be engineered to modify P-domainrecognition.

In some instances, a site-directed polypeptide comprises an amino acidsequence comprising at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid-cleaving domains (i.e., a HNH domain and RuvCdomain), and a modification, wherein prior to introduction of themodification the site-directed polypeptide is adapted to bind a firstPAM and after introduction of the modification, the site-directedpolypeptide is adapted to bind to a different PAM.

Modifications to Alter Nucleic Acid-Targeting Nucleic Acid Specificity

In some instances, a site-directed polypeptide can recognize a nucleicacid-targeting nucleic acid. A site-directed polypeptide can be modifiedto alter a nucleic acid-targeting nucleic acid specificity. For example,a site-directed polypeptide can be modified such that prior to themodifying the polypeptide targets a first a nucleic acid-targetingnucleic acid and after the modifying the site-directed polypeptidetargets a second a nucleic acid-targeting nucleic acid. In someinstances, altered nucleic acid-targeting nucleic acid specificity cancomprise a change in binding specificity (e.g., increased binding,decreased binding), and/or a change in the binding constant (e.g.,increase Kd, decrease Kd).

A site-directed polypeptide can be modified such that the site-directedpolypeptide can recognize a new type of a nucleic acid-targeting nucleicacid different from the the type the wild-type site-directed polypeptiderecognizes Any region of a site-directed polypeptide can be engineered(e.g., bridge helix, HNH and/or FINE-like domain, RuvC and/or RuvC-likedomain, basic patch) to alter PAM specificity according to the methodsof the disclosure.

In some instances, a site-directed polypeptide comprises an amino acidsequence comprising at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid-cleaving domains (i.e., a HNH domain and RuvCdomain), and a modification, wherein prior to introduction of themodification the site-directed polypeptide is adapted to bind a first anucleic acid-targeting nucleic acid and after introduction of themodification, the site-directed polypeptide is adapted to bind to adifferent a nucleic acid-targeting nucleic acid.

Modifications to Alter Hybridization Requirements

Insertions

A site-directed polypeptide can be modified to increase bindingspecificity to a target nucleic acid. A sequence may be inserted intothe site-directed polypeptide. In some instances, a HNH and/or HNH-likedomain may be inserted in a site-directed polypeptide. The non-nativesequence (e.g., HNH and/or FINE-like domain) may originate from anyspecies. The insertion may take place at any location in thesite-directed polypeptide. The insertion may occur in tandem (e.g.,adjacent) to the native HNH and/or HNH-like domain of the site-directedpolypeptide. The inserted HNH and/or HNH-like domain may comprise amutation. The inserted HNH and/or HNH-like domain may comprise amutation that reduces the nuclease activity of the domain. In someinstances, a RuvC and/or RuvC-like domain may be inserted in asite-directed polypeptide. The insertion may take place at any locationin the site-directed polypeptide. The insertion may occur in tandem(e.g., adjacent) to the native RuvC and/or RuvC-like domain of thesite-directed polypeptide. The inserted RuvC and/or RuvC-like domain maycomprise a mutation. The inserted RuvC and/or RuvC-like domain maycomprise a mutation that reduces the nuclease activity of the domain.

A site-directed polypeptide can be modified to increase bindingspecificity to a nucleic acid-targeting nucleic acid. A sequence may beinserted into the site-directed polypeptide. A HNH and/or HNH-likedomain may be inserted in a site-directed polypeptide. The non-nativesequence (e.g., HNH and/or HNH-like domain) may originate from anyspecies. The insertion may take place at any location in thesite-directed polypeptide. The insertion may occur in tandem (e.g.,adjacent) to the native HNH and/or FINE-like domain of the site-directedpolypeptide. The inserted HNH and/or HNH-like domain may comprise amutation. The inserted HNH and/or HNH-like domain may be comprise amutation that reduces the nuclease activity of the domain. A RuvC and/orRuvC-like domain may be inserted in a site-directed polypeptide. Theinsertion may take place at any location in the site-directedpolypeptide. The insertion may occur in tandem (e.g., adjacent) to thenative RuvC and/or RuvC-like domain of the site-directed polypeptide.The inserted RuvC and/or RuvC-like domain may comprise a mutation. Theinserted RuvC and/or RuvC-like domain may comprise a mutation thatreduces the nuclease activity of the domain.

A site-directed polypeptide can be engineered to comprise a polypeptidedomain that can bind to RNA-DNA hybrids (e.g., RNase domain, zinc fingerdomain). For example, a site-directed polypeptide can be engineered tocomprise an RNaseH domain. The inserted RNaseH domain may comprise amutation. The inserted RNaseH domain may be comprise a mutation thatreduces the nuclease activity of the domain.

A site-directed polypeptide can be engineered to comprise a polypeptidedomain that can bind to double-stranded DNA (e.g., domains comprisinghelix-turn-helix motifs, domains comprising leucine zipper motifs,domains comprising helix-loop-helix motifs, domains comprising zincfinger motifs). For example, a site-directed polypeptide can beengineered to comprise a helix-turn-helix motif. Non-limiting exemplaryhelix-turn-helix motifs include those from dnaB, TetR, MuB, P2R, CysB,BirA, the bacteriophage lambda repressor, Engrailed, Myb, LuxR, MarR,ETS, ZNF10a, Kox-1. The helix-loop-helix motif can be di-helical,tri-helical, tetrahelical, a winged helix-turn-helix, or other modifiedhelix-loop-helix. The inserted domain may be comprise a mutation. Theinserted domain may be comprise a mutation that reduces the nucleaseactivity of the domain.

Compensatory Mutations

A site-directed polypeptide can comprise a mutation and/or be engineeredsuch that it may preferentially bind to a mutated and/or engineerednucleic acid-targeting nucleic acid. Such mutation of a site-directedpolypeptide and nucleic acid-targeting nucleic acid pair can be referredto as a compensatory mutation. For example, a site-directed polypeptidecan be engineered such that its nuclease domain (e.g., HNH and/orFINE-like, RuvC and/or RuvC-like) is replaced by a nucleic acid bindingdomain (e.g., Csy4, Cas5, Cas6 nucleic acid binding domain). Asite-directed polypeptide can be engineered such that a nucleic acidbinding domain (e.g., Csy4, Cas5, Cas6 nucleic acid binding domain) isinserted into the site-directed polypeptide. The resulting site-directedpolypeptide can bind to a nucleic acid-targeting nucleic acid that ismutated and/or engineered to comprise a nucleic acid binding domainbinding site (e.g., binding site for Csy4, Cas5, Casio nucleic acidbinding domains). The nucleic acid-targeting nucleic acid can be mutatedand/or engineered to comprise a nucleic acid-binding domain binding sitein the minimum tracrRNA sequence. The nucleic acid-targeting nucleicacid can be mutated and/or engineered to comprise a nucleic acid bindingdomain binding site in the 3′ tracrRNA sequence. The nucleicacid-targeting nucleic acid can be mutated and/or engineered to comprisea nucleic acid binding domain binding site in the tracrRNA extension.

In some instances, a site-directed polypeptide comprises an amino acidsequence comprising at least 15% amino acid identity to a Cas9 from S.pyogenes, two nucleic acid-cleaving domains (i.e., a HNH domain and RuvCdomain), and a compensatory mutation, in which the site-directedpolypeptide is such that it can bind to an engineered nucleicacid-targeting nucleic acid but not an unmodified nucleic acid-targetingnucleic acid.

Methods to Generate Sticky Ends and Blunt Cuts

In some instances, one or more nickases (i.e., a site-directedpolypeptides comprising one substantially inactive nuclease domain) canbe used to generate targeted double stranded cuts in target nucleicacid. Each nickase of the one or more nickases can target one strand ofthe double-stranded target nucleic acid. In some instances, two nickasescan be used to generate a targeted double stranded cut.

The two nickases can cut the target nucleic acid generating a blunt endcut (wherein the cut sites of the target nucleic acid are the samelocation on each strand). The two nickases can cut the target nucleicacid at different locations within each strand such that some singlestranded nucleotides remain, thereby generating a sticky end.

Cleavage of target nucleic acid by two modified site-directedpolypeptides having nickase activity may be used to incur deletions orinsertions of nucleic acid material from a target nucleic acid bycleaving the target nucleic acid and allowing the cell to repair thesequence in the absence of an exogenously provided donor polynucleotide.In some instances, the methods of the disclosure can be used to knockout a gene. If a nucleic acid-targeting nucleic acid and two modifiedsite-directed polypeptides having nickase activity are co-administeredto cells with a donor polynucleotide sequence that includes at least asegment with homology to the target nucleic acid, new nucleic acidmaterial may be inserted/copied into the site. Such methods may be usedto add, i.e. insert or replace, nucleic acid material to a targetnucleic acid (e.g., to “knock in” a nucleic acid that encodes a protein,an siRNA, an miRNA, etc.), to add a tag (e.g., 6×His, a fluorescentprotein (e.g., a green fluorescent protein; a yellow fluorescentprotein, etc.), hemagglutinin (HA), FLAG, etc.), to add a regulatorysequence to a gene (e.g. promoter, polyadenylation signal, internalribosome entry sequence (IRES), 2A peptide, start codon, stop codon,splice signal, localization signal, etc.), to modify a nucleic acidsequence (e.g., introduce a mutation), and the like.

FIG. 32 depicts a method for generating blunt ends by nickases. Thetarget nucleic acid duplex 3210 can comprise a plurality of PAMsequences 3215 (boxed), wherein one PAM is on one strand of the targetnucleic acid 3210 and one PAM is on the other strand of the targetnucleic acid 3210. A nucleic acid-targeting nucleic acid 3205 as part ofa complex with the nickase (nickase not shown) can hybridize to thespacer sequence adjacent to the PAM 3215 on each strand of the targetnucleic acid 3210. The nickase can cleave one strand of the targetnucleic acid 3210. Cleavage is indicated by the triangles. If the PAMsare appropriately spaced the nickases can cut the target nucleic acid insubstantially the same place on each strand, thereby resulting in ablunt end. The PAM sequences may be separated by at least about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more nucleotides. The PAMsequences may be separated by at most about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, or 50 or more nucleotides. In some instances, the PAMsare spaced by 6 nucleotides (i.e., there are 6 nucleotides in betweeneach PAM). In some instances, the nucleic acid-targeting nucleic acidcleaves about 3 nucleotides 5′ of the PAM.

In some embodiments, two or more nickases can be employed to generatesticky ends. FIG. 33 illustrates how two nickases targeted tooverlapping regions on a target nucleic acid can result in a staggereddouble-stranded break resulting in sticky ends. The target nucleic acidduplex 3310 can comprise a plurality of PAM sequences 3315 (boxed). Anucleic acid-targeting nucleic acid 3305 as part of a complex with thenickase (nickase not shown) can hybridize to the spacer sequenceadjacent to the PAM 3315 on each strand of the target nucleic acid 3310.The nickase can cleave one strand of the target nucleic acid 3310.Cleavage is indicated by the triangles. If the PAMs are appropriatelyspaced the nickases can cut the target nucleic acid in staggeredlocations, thereby resulting in a sticky end. The PAM sequences may beseparated by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or morenucleotides. The PAM sequences may be separated by at most about 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. The distance of the PAMsequences can be related to the length of the sticky end generated. Forexample, the farther the PAMs are away from each other, the longer thesticky end will be.

A method for generating sticky ends using two or more nickases caninvolve PAM sequences substantially adjcent to one another (though onopposite strands). In some instances, the PAM sequences are separated byat least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. Insome instances, the PAM sequences are separated by at most about 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. In some instances, thePAM sequences are separated by one nucleotide. In some instances, thePAM sequences are separated by no nucleotides.

Methods for Enrichment and Sequencing of Target Nucleic Acids

General Overview

Sequencing can be useful for diagnosing disease by identifying mutationsand/or other sequence variants (e.g., polymorphisms). The methods of thedisclosure provide for methods, kits, and compositions for enriching atarget nucleic acid sequence without the use of amplificationmethodologies. A target nucleic acid can be enriched with the use of asite-directed polypeptide and a nucleic acid-targeting nucleic acid.

FIG. 3 depicts an exemplary embodiment of the methods of the disclosure.A site-directed polypeptide 305 can bind a nucleic acid-targetingnucleic acid 310, thereby forming a complex 306. The nucleicacid-targeting nucleic acid 310 can comprise a nucleic acid affinity tag311. The site-directed polypeptide 305 can comprise a nuclease domain.The site-directed polypeptide 305 can be enzymatically active. Thesite-directed polypeptide 305 can comprise an affinity tag 315. Thenucleic acid-targeting nucleic acid 310 can hybridize to a targetnucleic acid 320. In some embodiments, a plurality of complexes 306 canhybridize to a plurality of locations within a target nucleic acid 320.In a cleavage step 325, the nuclease domain of a site-directedpolypeptide 305 can cleave, or cut 330 the target nucleic acid 320. Theexcised target nucleic acid 340 can be purified in a purification step335. Adaptors 345 can be ligated to the excised target nucleic acid. Theadaptors can facilitate sequencing of the excised target nucleic acid.

FIG. 4 depicts an exemplary embodiment of the methods of the disclosure.A site-directed polypeptide 405 can interact with a nucleicacid-targeting nucleic acid 410, thereby forming a complex 406. Thesite-directed polypeptide 405 can comprise a nuclease domain. In someembodiments, the nuclease domain of the site-directed polypeptide 405can be enzymatically inactive. The site-directed polypeptide 405 cancomprise an affinity tag 415. The nucleic acid-targeting nucleic acid410 can hybridize to a target nucleic acid 420. The nucleicacid-targeting nucleic acid 410 can comprise a nucleic acid affinity tag411. The affinity tag 411 of the nucleic acid-targeting nucleic acid cancomprise a hairpin structure. A plurality of complexes 406 can hybridizeto a plurality of locations within a target nucleic acid 420. In afragmenting step 225, the target nucleic acid 420 can be fragmented intotarget nucleic acid fragment 445 (also herein referred to as a “targetnucleic acid”). The site-directed polypeptide 405 can be purified by acapture agent 440 that can bind to the affinity tag 415 of thesite-directed polypeptide 405. The fragmented target nucleic acid 445can be eluted from the complex 406 in a purification step 450. In thesame step, or optionally, in a different step, adaptors 455 can beligated to the target nucleic acid. The adaptors can facilitatesequencing of the target nucleic acid.

Complex of a Nucleic Acid-Targeting Nucleic Acid and a Site-DirectedPolypeptide

A nucleic acid-targeting nucleic acid can interact with a site-directedpolypeptide (e.g., a nucleic acid-guided nuclease, e.g. Cas9), therebyforming a complex. The nucleic acid-targeting nucleic acid can guide thesite-directed polypeptide to a target nucleic acid.

In some embodiments, a nucleic acid-targeting nucleic acid can beengineered such that the complex (e.g., comprising a site-directedpolypeptide and a nucleic acid-targeting nucleic acid) can bind outsideof the cleavage site of the site-directed polpeptide. In this case, thetarget nucleic acid may not interact with the complex and the targetnucleic acid can be excised (e.g., free from the complex).

In some embodiments, a nucleic acid-targeting nucleic acid can beengineered such that the complex can bind inside of the cleavage site ofthe site-directed polpeptide. In this case, the target nucleic acid caninteract with the complex and the target nucleic acid can be bound(e.g., bound to the complex).

The nucleic acid-targeting nucleic acid can be engineered in such a waythat the complex (e.g., comprising a site-directed polypeptide and/or anucleic acid-targeting nucleic acid) can hybridize to a plurality oflocations within a nucleic acid sample.

A plurality of complexes can be contacted to a nucleic acid sample. Theplurality of complexes can comprise nucleic acid-targeting nucleic acidsengineered to hybridize to the same sequence. The plurality of complexescan comprise nucleic acid-targeting nucleic acids engineered tohybridize to the different sequences.

The sequences can be at different locations within a target nucleicacid. The locations can comprise the same, or similar, target nucleicacid sequences. The locations can comprise different target nucleic acidsequences. The locations can be a defined distance from each other. Thelocations can be less than 10 kilobases (Kb) apart, less than 8 Kbapart, less than 6 Kb apart, less than 4 Kb apart, less than 2 Kb apart,less than 1 Kb apart, less than 900 nucleotides apart, less than 800nucleotides apart, less than 700 nucleotides apart, less than 600nucleotides apart, less than 500 nucleotides apart, less than 400nucleotides apart, less than 300 nucleotides apart, less than 200nucleotides apart, less than 100 nucleotides apart.

The complexes can cleave the target nucleic acid which can result in anexcised target nucleic acid that can be less than 10 kilobases (Kb)long, less than 8 Kb long, less than 6 Kb long, less than 4 Kb long,less than 2 Kb long, less than 1 Kb long, less than 900 nucleotideslong, less than 800 nucleotides long, less than 700 nucleotides long,less than 600 nucleotides long, less than 500 nucleotides long, lessthan 400 nucleotides long, less than 300 nucleotides long, less than 200nucleotides long, less than 100 nucleotides long.

The complexes can be bound to a fragmented target nucleic acid that canbe be less than 10 kilobases (Kb) long, less than 8 Kb long, less than 6Kb long, less than 4 Kb long, less than 2 Kb long, less than 1 Kb long,less than 900 nucleotides long, less than 800 nucleotides long, lessthan 700 nucleotides long, less than 600 nucleotides long, less than 500nucleotides long, less than 400 nucleotides long, less than 300nucleotides long, less than 200 nucleotides long, less than 100nucleotides long.

Methods for Detecting Off-Target Binding Sites of Site-DirectedPolypeptides

General Overview

This disclosure describes methods, compositions, systems, and/or kitsfor determining off target binding sites of site-directed polypeptides.In some embodiments of the disclosure a site-directed polypeptide cancomprise a nucleic acid-targeting nucleic acid, thereby forming acomplex. The complex can be contacted with a target nucleic acid. Thetarget nucleic acid can be captured with capture agents that can bind tothe affinity tags of the complex. The identity of the target nucleicacid can be determined through sequencing. Sequencing (e.g., highthroughput sequencing, e.g., Illumina, Ion Torrent) can also identifythe frequency of off-target binding sites of the site-directedpolypeptide and/or complex, by counting the number of times a particularbinding site is read. The methods, compositions, systems, and/or kits ofthe disclosure can facilitate the development of more accurately andspecifically targeted site-directed polypeptides.

FIG. 5 depicts an exemplary embodiment of the methods of the disclosure.A site-directed polypeptide 505 can comprise an affinity tag 510. Thesite-directed polypeptide can comprise a nucleic acid-binding domain515. The nucleic acid-binding domain 515 can be a nucleic acid. In someembodiments, the nucleic acid-binding domain 515, and the site-directedpolypeptide 505 form a complex 531. The complex 131 can be contacted 525with a target nucleic acid 530. In a preferred embodiment, the targetnucleic acid 530 is DNA (e.g. genomic DNA or gDNA). The complex can beaffinity purified 535 with a capture agent 540. The capture agent 540can bind to the affinity tag 510 from the site-directed polypeptide 505.The capture agent 540 can comprise a second affinity tag 545. Thecapture agent 540 can be affinity purified 550 by binding to a solidsupport 555. In some embodiments, the solid support 555 is a bead coatedwith an affinity reagent that can bind to the affinity tag 545 of thecapture agent. Optionally, the solid support 555 can bind to theaffinity tag 510 of the site-directed polypeptide 505 to facilitatepurification. In some embodiments, one or more rounds of purificationcan occur. Each round can comprise contacting a solid support 555 withthe affinity tags of the site-directed polypeptide 510 and/or thecapture agent 545. The affinity purified complex can be eluted from thetarget nucleic acid 530. The target nucleic acid can subsequently beprepared for further processing. Processing can include downstreamanalysis methods, e.g. sequencing.

FIG. 6 depicts an exemplary embodiment of the methods of the disclosure.A site-directed polypeptide 605 can comprise an affinity tag 610. Thesite-directed polypeptide 605 can comprise a nucleic acid-binding domain615. The nucleic acid-binding domain 615 can be a nucleic acid. In someembodiments, the nucleic acid-binding domain 615 can comprise anaffinity tag 620. In some embodiments, the nucleic acid-binding domain615 and the site-specific polypeptide 605 can form a complex 631. Thecomplex 631 may be contacted 625 with a target nucleic acid 630. In apreferred embodiment, the target nucleic acid 630 is DNA. The complex631 can be affinity purified 635 with a capture agent 640. The captureagent 640 may be a conditionally enzymatically inactive site-directedpolypeptide. The capture agent 640 can be a conditionally enzymaticallyinactive variant of Csy4. The capture agent 640 can bind to the affinitytag 620. The capture agent 640 can comprise an affinity tag 645. Thecapture agent 640 can be affinity purified 650 by binding to a solidsupport 655. In some embodiments, the solid support is a bead coatedwith an affinity reagent that can bind to the affinity tag 645 of thecapture agent 640. Optionally, the solid support 655 can bind to theaffinity tag 610 of the site-directed polypeptide 605 to facilitatepurification. In some embodiments, two rounds of purification can occur,each comprising contacting a solid support 655 with the affinity tags ofthe site-directed polypeptide 610 and/or the capture agent 640. Cleavageof the affinity tag 620 can facilitate elution 660 of the target nucleicacid 630 from the solid support 655. The target nucleic acid 630 cansubsequently be prepared for further downstream analysis methods such assequencing.

Methods

The disclosure provides methods for nuclease immunoprecipitation andsequencing (NIP-Seq). In some embodiments, the method can comprise a)contacting a nucleic acid sample with a complex comprising anenzymatically inactive site-directed polypeptide, a site-directedpolypeptide, and a nucleic acid-targeting nucleic acid. The complex canhybridize to a target nucleic acid. The complex can be captured with acapture agent, and the target nucleic acid bound to the complex can besequenced. In some embodiments, the method can further comprisedetermining the identity of the off-target binding site. The method canbe performed using any of the site-directed polypeptides, nucleicacid-targeting nucleic acids, and complexes of site-directedpolypeptides and nucleic acid-targeting nucleic acids as describedherein.

The methods can be performed outside of a cell. For example, a samplecan comprise purified genomic DNA, cell lysate, homogenized tissue,plasma, and the like. The methods can be performed in cells.

The site-directed polypeptide-target nucleic acid complexes can be fixedor cross-linked to form complexes. The cells can be crosslinked beforethey are lysed. Fixed or cross-linking cells can stabilize protein-DNAcomplexes in the cell. Suitable fixatives and cross-linkers can include,formaldehyde, glutaraldehyde, ethanol-based fixatives, methanol-basedfixatives, acetone, acetic acid, osmium tetraoxide, potassiumdichromate, chromic acid, potassium permanganate, mercurials, picrates,formalin, paraformaldehyde, amine-reactive NHS-ester crosslinkers suchas bis[sulfosuccinimidyl] suberate (BS3),3,3′-dithiobis[sulfosuccinimidylpropionate] (DTSSP), ethylene glycolbis[sulfosuccinimidylsuccinate (sulfo-EGS), disuccinimidyl glutarate(DSG), dithiobis[succinimidyl propionate] (DSP), disuccinimidyl suberate(DSS), ethylene glycol bis[succinimidylsuccinate] (EGS),NHS-ester/diazirine crosslinkers such as NHS-diazirine,NHS-LC-diazirine, NHS-SS-diazirine, sulfo-NHS-diazirine,sulfo-NHS-LC-diazirine, and sulfo-NHS-SS-diazirine.

The nucleic acid (e.g., genomic DNA) can be treated to fragment the DNAbefore affinity purification. Fragmentation can be performed throughphysical, mechanical or enzymatic methods. Physical fragmentation caninclude exposing a target polynucleotide to heat or to ultraviolet (UV)light. Mechanical disruption may be used to mechanically shear a targetpolynucleotide into fragments of the desired range. Mechanical shearingmay be accomplished through a number of methods known in the art,including repetitive pipetting of the target polynucleotide, sonicationand nebulization. Target polynucleotides may also be fragmented usingenzymatic methods. In some cases, enzymatic digestion may be performedusing enzymes such as using restriction enzymes. Restriction enzymes maybe used to perform specific or non-specific fragmentation of targetpolynucleotides. The methods may use one or more types of restrictionenzymes, generally described as Type I enzymes, Type II enzymes, and/orType III enzymes. Type II and Type III enzymes are generallycommercially available and well known in the art. Type II and Type IIIenzymes recognize specific sequences of nucleotide nucleotides within adouble stranded polynucleotide sequence (a “recognition sequence” or“recognition site”). Upon binding and recognition of these sequences,Type II and Type III enzymes cleave the polynucleotide sequence. In somecases, cleavage will result in a polynucleotide fragment with a portionof overhanging single stranded DNA, called a “sticky end.” In othercases, cleavage will not result in a fragment with an overhang, creatinga “blunt end.” The methods may comprise use of restriction enzymes thatgenerate either sticky ends or blunt ends. Fragments of nucleic acidscan also be generated via amplification techniques (e.g. polymerasechain reaction, long range polymerase chain reaction, linear polymerasechain reaction, and etc.).

Once fragmented, the complexes comprising the site-directed polypeptidecan be purified by incubation with a solid support. For example, if thesite-directed polypeptide comprises a biotin tag, the solid support canbe coated with avidin or streptavidin to bind to the biotin tag.

In some embodiments, once fragmented, the complexes comprising thesite-directed polypeptide, the target nucleic acid, and/or the nucleicacid-targeting nucleic acid are purified by incubation with a captureagent. A capture agent can refer to any agent that can bind to anaffinity tag fused to the site-directed polypeptide. Exemplary captureagents can include, biotin, streptavidin, and antibodies. For example,if the affinity tag fused to the site-directed polypeptide is a FLAGtag, then the capture agent will be an anti-FLAG-tag antibody. In someembodiments, the capture agent can comprise an affinity tag (e.g.,biotin, streptavidin).

In some instances, the capture agent is an enzymatically inactiveendoribonuclease. For example, a capture agent can be an enzymaticallyinactive site-directed polypeptide, an enzymatically inactive Csy4,Cas5, or Cas6.

The capture agent can be purified with a solid support. For example, ifthe capture agent comprises a biotin tag, the bead can be coated withavidin or streptavidin to bind the biotinylated capture agent.

In some embodiments of the method, two or more rounds of purificationcan be performed. At least 1, 2, 3, 4, 5, 6, 7 or more rounds ofpurification can be performed. At most 1, 2, 3, 4, 5, 6, 7 or morerounds of purification can be performed. A first round of purificationcan comprise purification with a solid support that can bind to theaffinity tag of the capture agent and a second round of purification cancomprise purification with a solid support that can bind to the affinitytag of the site-directed polypeptide. A first round of purification cancomprise purification with a solid support that can bind to the affinitytag of the site-directed polypeptide and a second round of purificationcan comprise purification with a solid support that will bind to theaffinity tag of the capture agent. The method can be used to optimizethe binding specificity of a site-directed polypeptide by performing themethod more than once.

The captured complex can comprise site-directed polypeptide and a targetnucleic acid. The target nucleic acid can be eluted from thesite-directed polypeptide complex by methods such as high salt washing,ethanol precipitation, boiling, and gel purification.

The eluted DNA can be prepared for sequencing analysis (e.g., shearing,ligation of adaptors). Preparation for sequencing analysis can includethe generation of sequencing libraries of the eluted target nucleicacid. Sequencing analysis can determine the identity and frequency ofoff-target binding sites of site-directed polypeptides. Sequencedetermination will also be performed using methods that determine many(typically thousands to billions) nucleic acid sequences in anintrinsically parallel manner, where many sequences are read outpreferably in parallel using a high throughput serial process. Suchmethods include but are not limited to pyrosequencing (for example, ascommercialized by 454 Life Sciences, Inc., Branford, Conn.); sequencingby ligation (for example, as commercialized in the SOLiD™ technology,Life Technology, Inc., Carlsbad, Calif.); sequencing by synthesis usingmodified nucleotides (such as commercialized in TruSeq™ and HiSeg™technology by Illumina, Inc., San Diego, Calif., HeliScope™ by HelicosBiosciences Corporation, Cambridge, Mass., and PacBio RS by PacificBiosciences of California, Inc., Menlo Park, Calif.), sequencing by iondetection technologies (Ion Torrent, Inc., South San Francisco, Calif.);sequencing of DNA nanoballs (Complete Genomics, Inc., Mountain View,Calif.); nanopore-based sequencing technologies (for example, asdeveloped by Oxford Nanopore Technologies, LTD, Oxford, UK), and otherknown highly parallelized sequencing methods.

In some embodiments, the method further comprises collecting data andstoring data. The data can be machine readable and can be stored and/orcollected on a computer server (e.g. FIG. 31 and Example 27).

Methods for Detecting Sequence Variants in Nucleic Acids

General Overview

In some embodiments, the methods of the disclosure provide for detectingsequence variants in nucleic acids. The method can be performed usingany of the site-directed polypeptides, nucleic acid-targeting nucleicacids, and complexes of site-directed polypeptides and nucleicacid-targeting nucleic acids as described herein. As depicted in FIG. 7,a nucleic acid sample 705 can be ligated 720 with a nucleic acid tag710. The nucleic acid tag can be a single guide RNA. The nucleic acidtag can comprise a crRNA. The nucleic acid tag can comprise a detectablelabel 715. Together, the nucleic acid sample 705 ligated to the nucleiacid tag 710 can be referred to as a tagged test sample 721. The taggedtest sample 721 can be contacted 725 to an array 740 comprisingimmobilized oligonucleotides 735. The immobilized oligonucleotides 735can be referred to as a nucleic acid library. The oligonucleotides 735can be double stranded DNA. The oligonucleotides 735 can comprise adetectable label 730. The individual members of the tagged test sample721 can hybridize 745 to the oligonucleotides 735 to which they shareenough complementarity to facilitate hybridization. The amount ofhybridization can be quantified by comparing the intensities of the twodetectable labels 715 and 730. For example, hybridized oligonucleotidescan display two detectable labels. Unhybridized oligonucleotides candisplay one detectable label 730. The hybridized sample can be contactedwith a site-directed polypeptide 750. The site-directed polypeptide cancleave 755 the oligonucleotides 735 in the array 740 that havehybridized with members of the tagged test sample 721. Cleavage by thesite-directed polypeptide can allow the hybridized members of the taggedtest sample 721 to be removed. After cleavage by the site-directedpolypeptide 750, only unhybridized oligonucleotide detectable labels 760will remain on the array. The remaining detectable label 760 can bequantified. The quantification of the remaining detectable labels 760can be correlated to which sequences were represented in the nucleicacid sample 705 and which were not. Oligonucleotides that do not displaya remaining detectable label 760 correspond to sequences that wererepresented in the nucleic acid sample 705. Oligonucleotides thatdisplay a remaining detectable label 760 correspond to sequences thatwere not represented in the nucleic acid sample 705.

In some embodiments, a nucleic acid sample 805 can be ligated 820 with anucleic acid tag 810. The nucleic acid tag can be a single guide RNA.The nucleic acid tag can comprise a crRNA. The nucleic acid tag cancomprise a detectable label 815. Together, the nucleic acid sampleligated to the nuclei acid tag can be referred to as a tagged testsample 821. The tagged test sample 821 can be contacted 825 to an array840 comprising immobilized oligonucleotides 835. The immobilizedoligonucleotides can be referred to as a nucleic acid library. Theoligonucleotides 835 can be double stranded DNA. The individual membersof the tagged test sample 821 can hybridize 845 to the oligonucleotides835 to which they share enough complementarity to facilitatehybridization. The hybridized sample can be contacted with asite-directed polypeptide 850. The site-directed polypeptide can cleave855 the oligonucleotides 835 in the array 840 that have hybridized withmembers of the tagged test sample 821. Cleavage by the site-directedpolypeptide 850 can allow the hybridized members of the tagged testsample 821 to be removed. Cleavage by the site-directed polypeptide 850can allow a portion of the immobilized olignucleotide to be cleaved andseparated from the array 860. The separated cleaved oligonucleotides 860can be ligated 865 to appropriate adaptors 870 for sequencing.Sequencing of the cleaved oligonucleotides 860 can determine thesequences represented in the nucleic acid sample 805.

In some embodiments, a nucleic acid library can be generated forsequencing analysis using commercially available high throughputsequencing platforms. The library can comprise nucleic acids that cancomprise one or more sequencing tags 930 and a target sequence 945. Thetarget sequence 945 can be a sequence that may be represented in anucleic acid sample 905. A target sequence 945 can comprise aprotospacer adjacent motif (PAM) sequence. Optionally, nucleic acids ina nucleic acid library can comprise one or more identifyingpolynucleotide sequences 935, and one or more extension sequences 940.In this embodiment, a nucleic acid sample 905 can be ligated 920 with anucleic acid tag 910. The nucleic acid tag can be a single guide RNA.The nucleic acid tag can comprise a crRNA. Optionally, the nucleic acidtag can comprise an affinity tag 915. Together, the nucleic acid sampleligated to the nuclei acid tag can be referred to as a tagged testsample 921. The tagged test sample 921 can be contacted 925 to a nucleicacid library. The tagged test sample 921 can hybridize to a nucleic acidin the nucleic acid library, forming a complex 946. The hybridizedtagged test sample and nucleic acid library can be contacted with asite-directed polypeptide 950. The site-directed polypeptide 950 cancleave the hybridized nucleic acid library members. The cleaved nucleicacid library members 965 can be separated from the uncleaved members.The uncleaved members can be subjected to sequencing analysis.Sequencing analysis can determine which sequences were represented inthe nucleic acid sample 905. For example, the sequences of the uncleavedmembers can correspond to sequences that were not represented in thenucleic acid sample 905. These sequences can be removed from the knownsequences in the nucleic acid library. The resulting sequences can bethe sequences of the cleaved members 965 of the nucleic acid librarywhich can correspond to sequences that were represented in the nucleicacid sample 905.

The site-directed polypeptide 950 can comprise an affinity tag 955.Optionally, the site-directed polypeptide 950 can be an enzymaticallyinactive variant of a site-directed polypeptide. In some embodiments, anenzymatically inactive site-directed polypeptide can be contacted to ahybridized nucleic acid library (e.g., complex 946). The site-directedpolypeptide can bind but cannot cleave the hybridized nucleic acidlibrary members. The site-directed polypeptide can be affinity purified970 with a capture agent 975 that can bind to the affinity tag 955.Optionally, the complex 946 can be affinity purified with a captureagent that can bind to the affinity tag 915. The affinity purifiednucleic acid library members can be subjected to sequencing analysis. Inthis embodiment, the sequenced nucleic acid library members cancorrespond to sequences that are represented in the nucleic acid sample905.

Sequencing

Methods for detecting sequence variants can comprise sequencing thevariants. Sequence determination can be performed using methods thatdetermine many (typically thousands to billions) nucleic acid sequencesin an intrinsically parallel manner, where many sequences are read outpreferably in parallel using a high throughput serial process. Suchmethods can include but are not limited to pyrosequencing (for example,as commercialized by 454 Life Sciences, Inc., Branford, Conn.);sequencing by ligation (for example, as commercialized in the SOLiD™technology, Life Technology, Inc., Carlsbad, Calif.); sequencing bysynthesis using modified nucleotides (such as commercialized in TruSeq™and HiSeg™ technology by Illumina, Inc., San Diego, Calif., HeliScope™by Helicos Biosciences Corporation, Cambridge, Mass., and PacBio RS byPacific Biosciences of California, Inc., Menlo Park, Calif.), sequencingby ion detection technologies (Ion Torrent, Inc., South San Francisco,Calif.); sequencing of DNA nanoballs (Complete Genomics, Inc., MountainView, Calif.); nanopore-based sequencing technologies (for example, asdeveloped by Oxford Nanopore Technologies, LTD, Oxford, UK), capillarysequencing (e.g, such as commercialized in MegaBACE by MolecularDynamics), electronic sequencing, single molecule sequencing (e.g., suchas commercialized in SMRT™ technology by Pacific Biosciences, MenloPark, Calif.), droplet microfluidic sequencing, sequencing byhybridization (such as commercialized by Affymetrix, Santa Clara,Calif.), bisulfate sequencing, and other known highly parallelizedsequencing methods.

Real Time PCR

Methods for detecting sequence variants can comprise detecting thevariants using real time PCR. Sequence determination can be performed byreal time polymerase chain reaction (RT-PCR, also referred to asquantitative-PCR (QPCR)) can detect an amount of amplifiable nucleicacid present in a sample. QPCR is a technique based on the polymerasechain reaction, and can be used to amplify and simultaneously quantify atarget nucleic acid. QPCR can allow for both detection andquantification of a specific sequence in a target nucleic acid sample.The procedure can follow the general principle of polymerase chainreaction, with the additional feature that the amplified target nucleicacid can be quantified as it accumulates in the reaction in real timeafter each amplification cycle. Two methods of quantification can be:(1) use of fluorescent dyes that intercalate with double-stranded targetnucleic acid, and (2) modified DNA oligonucleotide probes that fluorescewhen hybridized with a complementary target nucleic acid. In the firstmethod, a target nucleic acid-binding dye can bind to alldouble-stranded (ds) nucleic acid in PCR, resulting in fluorescence ofthe dye. An increase in nucleic acid product during PCR therefore canlead to an increase in fluorescence intensity and can be measured ateach cycle, thus allowing nucleic acid concentrations to be quantified.The reaction can be prepared similarly to a standard PCR reaction, withthe addition of fluorescent (ds) nucleic acid dye. The reaction can berun in a thermocycler, and after each cycle, the levels of fluorescencecan be measured with a detector; the dye can only fluoresce when boundto the (ds)nucleic acid (i.e., the PCR product). With reference to astandard dilution, the (ds) nucleic acid concentration in the PCR can bedetermined. The values obtained can not have absolute units associatedwith it. A comparison of a measured DNA/RNA sample to a standarddilution can give a fraction or ratio of the sample relative to thestandard, allowing relative comparisons between different tissues orexperimental conditions. To ensure accuracy in the quantification, theexpression of a target gene can be normalized to a stably expressedgene. This can allow for correction of possible differences in nucleicacid quantity or quality across samples. The second method can use asequence-specific RNA or DNA-based probe to quantify only the nucleicacid containing the probe sequence; therefore, use of the reporter probecan increase specificity, and can allow quantification even in thepresence of some non-specific nucleic acid amplification. This can allowfor multiplexing, (i.e., assaying for several genes in the same reactionby using specific probes with differently colored labels), provided thatall genes are amplified with similar efficiency. This method can becarried out with a nucleic acid-based probe with a fluorescent reporter(e.g. 6-carboxyfluorescein) at one end and a quencher (e.g.,6-carboxy-tetramethylrhodamine) of fluorescence at the opposite end ofthe probe. The close proximity of the reporter to the quencher canprevent detection of its fluorescence. Breakdown of the probe by the 5′to 3′ exonuclease activity of a polymerase (e.g., Taq polymerase) canbreak the reporter-quencher proximity and thus can allow unquenchedemission of fluorescence, which can be detected. An increase in theproduct targeted by the reporter probe at each PCR cycle can result in aproportional increase in fluorescence due to breakdown of the probe andrelease of the reporter

The reaction can be prepared similarly to a standard PCR reaction, andthe reporter probe can be added. As the reaction commences, during theannealing stage of the PCR both probe and primers can anneal to thetarget nucleic acid. Polymerization of a new DNA strand can be initiatedfrom the primers, and once the polymerase reaches the probe, its5′-3′-exonuclease can degrade the probe, physically separating thefluorescent reporter from the quencher, resulting in an increase influorescence. Fluorescence can be detected and measured in a real-timePCR thermocycler, and geometric increase of fluorescence can correspondto exponential increase of the product is used to determine thethreshold cycle in each reaction. Relative concentrations of DNA presentduring the exponential phase of the reaction can be determined byplotting fluorescence against cycle number on a logarithmic scale (so anexponentially increasing quantity can give a straight line). A thresholdfor detection of fluorescence above background can be determined. Thecycle at which the fluorescence from a sample crosses the threshold canbe called the cycle threshold, Ct. Since the quantity of DNA can doubleevery cycle during the exponential phase, relative amounts of DNA can becalculated, (e.g. a sample with a Ct of 3 cycles earlier than anotherhas 23=8 times more template). Amounts of nucleic acid (e.g., RNA orDNA) can be determined by comparing the results to a standard curveproduced by a real-time PCR of serial dilutions (e.g. undiluted, 1:4,1:16, 1:64) of a known amount of nucleic acid. The QPCR reaction caninvolve a dual fluorophore approach that takes advantage of fluorescenceresonance energy transfer (FRET), (e.g., LIGHTCYCLER hybridizationprobes, where two oligonucleotide probes can anneal to the amplicon).The oligonucleotides can be designed to hybridize in a head-to-tailorientation with the fluorophores separated at a distance that iscompatible with efficient energy transfer. Other examples of labeledoligonucleotides that are structured to emit a signal when bound to anucleic acid or incorporated into an extension product include:SCORPIONS probes, Sunrise (or AMPLIFLOUR) primers, and LUX primers andMOLECULAR BEACONS probes. The QPCR reaction can use fluorescent Taqmanmethodology and an instrument capable of measuring fluorescence in realtime (e.g., ABI Prism 7700 Sequence Detector). The Taqman reaction canuse a hybridization probe labeled with two different fluorescent dyes.One dye can be a reporter dye (6-carboxyfluorescein), the other can be aquenching dye (6-carboxy-tetramethylrhodamine). When the probe isintact, fluorescent energy transfer can occur and the reporter dyefluorescent emission can be absorbed by the quenching dye. During theextension phase of the PCR cycle, the fluorescent hybridization probecan be cleaved by the 5′-3′ nucleolytic activity of the DNA polymerase.On cleavage of the probe, the reporter dye emission can no longertransferred efficiently to the quenching dye, resulting in an increaseof the reporter dye fluorescent emission spectra. Any nucleic acidquantification method, including real-time methods or single-pointdetection methods can be use to quantify the amount of nucleic acid inthe sample. The detection can be performed several differentmethodologies (e.g., staining, hybridization with a labeled probe;incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of 32P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment. Thequantification can or can not include an amplification step. Thequantitation can not be experimental.

Microarray

Methods for detecting sequence variants can comprise sequencing and/ordetecting the variants using a microarray. Microarrays can be used fordetermining the expression level of a plurality of genes in a nucleicacid sample. Microarrays can be used for determining sequence identityof a plurality of sequences in a nucleic acid sample.

A microarray can comprise a substrate. Substrates can include, but arenot limited to, glass and modified or functionalized glass, plastics(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes,Teflon™, and the like), polysaccharides, nylon or nitrocellulose,resins, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, and plastics.

Microarrays can comprise a plurality of polynucleotide probes. Amicroarray can comprise about 1, 10, 100, 1000, 5000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 110000, 120000or more probes.

Probes can be can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140 nucleotides or more in length.

In some embodiments, probes can comprise sequence information for aspecific set of genes and/or species. A probe can be complementary to anucleic acid sequence encoding a host protein. A probe can becomplementary to a non-coding nucleic acid sequence. A probe can becomplementary to a DNA sequence. A probe can be complementary to an RNAsequence.

Probes can be immobilized on a microarray. The immobilization ofpolynucleotides on a solid substrate can be achieved by direct synthesis(e.g., photolithographic synthesis) of polynucleotides on a solidsubstrate or by immobilization (spotting) of previously synthesizedpolynucleotides on predetermined regions of a solid substrate.Polynucleotides can be immobilized on a microarray substrate byactivating a surface of a solid substrate with a nucleophilic functionalgroup (e.g., an amino group), coupling biomolecules (e.g.,polynucleotides) activated with a good leaving group to thesurface-activated solid substrate, and removing unreacted reactants.Probes can be immobilized to a bead further conjugated through acovalent or ionic attachment to a solid support. Probes can beimmobilized onto a substrate using a specific film having a lowconductivity and a low melting temperature, namely a gold film. Anapplied electromagnetic radiation can melt and can ablate the film atthe impingement site. The film can be in contact with a colloidaldispersion and upon melting can generate a convective flow at thereaction site, thereby leading to adhering of an insoluble particle inthe dispersion to the specifically melted site.

A microarray can analyze a nucleic acid sample comprising nucleic acidsof unknown identity (e.g., test sample) by comparing the nucleic acidsample of unknown identity with a reference sample. A nucleic acidsample can be prepared from DNA (e.g., isolated DNA, genomic DNA,extrachromasomal DNA). A nucleic acid sample can be prepared from RNA.RNA can be reverse transcribed into DNA with a gene-specific primer or auniversal primer. The reverse transcribed DNA (e.g., cDNA), can betreated with Rnase or base (e.g., NaOH) to hydrolyze the RNA. The cDNAcan be labelled with a dye (e.g, Cy3, Cy5) with N-hydroxysuccinimidechemistry or similar labeling chemistries. Suitable fluorescent dyes caninclude a variety of commercial dyes and dye derivatives such as thosethat are denoted Alexa, Fluorescein, Rhodamine, FAM, TAMRA, Joe, ROX,Texas Red, BODIPY, FITC, Oregon Green, Lissamine and others. Thereference sample can be labeled with a different dye than the testsample.

The test sample and the reference sample can be applied to a microarrayto contact multiple spots simultaneously. The test sample and thereference sample can be applied to the microarray under hybridizingconditions that can allow the nucleic acids in the nucleic acid sampleto bind to a complement probe on the microarray. Various reaction stepscan be performed with the bound molecules in the microarray, includingexposure of bound reactant molecules to washing steps. The progress oroutcome of the reaction can be monitored at each spot (e.g., probe) inthe microarray in order to characterize the nucleic acid sampleimmobilized on the chip. Microarray analysis usually can require anincubation period that can range from minutes to hours. The duration ofthe incubation period can be assay dependent and can be determined by avariety of factors, such as the type of reactant, degree of mixing,sample volume, target copy number, and density of the array. During theincubation period, nucleic acids in the nucleic acid sample can be inintimate contact with the microarray probes.

Detection can be performed using a confocal scanning instrument withlaser excitation and photomultiplier tube detection, such as theScanArray 3000 provided by GSI Lumonics (Billerica, Mass.). Confocal andnon-confocal fluorescent detection systems can be used to implement themethod such as those provided by Axon Instruments (Foster City, Calif.),Genetic MicroSystems (Santa Clara, Calif.), Molecular Dynamics(Sunnyvale, Calif.) and Virtek (Woburn, Mass.). Alternative detectionsystems can include scanning systems that use gas, diode and solid statelasers as well as those that use a variety of other types ofillumination sources such as xenon and halogen bulbs. In addition tophotomultiplier tubes, detectors can include cameras that use chargecoupled device (CCD) and complementary metal oxide silicon (CMOS) chips.

The ratio of the intensities of the two dyes from the test sample andthe reference sample can be compared for each probe. The strength of thesignal detected from a given microarray spot can be directlyproportional to the degree of hybridization of a nucleic acid in thesample to the probe at a given spot (e.g., a spot comprises a probe).Analysis of the fluorescence intensities of hybridized microarrays caninclude spot segmentation, background determination (and possiblesubtraction), elimination of bad spots, followed by a method ofnormalization to correct for any remaining noise. Normalizationtechniques can include global normalization on all spots or a subset ofthe spots such as housekeeping genes, prelog shifting to obtain betterbaseline matches, or in the case of two (or more) channel hybridizationsfinding the best fit that helps to give an M vs. A plot that is centeredabout M=0 and/or that helps to give a log(Red) vs. log(Green) plot thatis centered about the diagonal with the smallest spread. The M vs. Aplot can also be referred to as the R vs. I plot, where R is a ratio,such as R=log₂(Red/Green) and I is an intensity, such as I=logVRed*Green. Scaling, shifting, best fits through scatter plots, etc. canbe techniques utilized to normalize microarray datasets and to givebetter footing for subsequent analysis. Most of these normalizationmethods can have some underlying hypothesis behind them (such as “mostgenes within the study do not vary much”).

Tagged Nucleic Acids and Methods of Use

General Overview

The disclosure provides for kits, methods, and compositions for taggednucleic acid-targeting nucleic acids, as described herein. FIG. 10depicts an exemplary embodiment of nucleic acid-targeting nucleic acid1005 of the disclosure. A nucleic acid-targeting nucleic acid cancomprise one or more non-native sequences (e.g., tags) 1010/1015. Anucleic acid-targeting nucleic acid can comprise a non-native sequence1010/1015 at either the 3′ end, the 5′ end, or both the 3′ and 5′ end ofthe nucleic acid-targeting nucleic acid.

In some instances, a nucleic acid-targeting nucleic acid can be nucleicacid-targeting nucleic acids as described herein, and comprise one ormore non-native sequences, such as either at the 3′end, the 5′ end orboth the 3′ and 5′ ends of the nucleic acid-targeting nucleic acid.

In some instances, the nucleic acid-targeting nucleic acid is adouble-guide nucleic acid-targeting nucleic acid as described herein,and as depicted in FIG. 11. A double-guide nucleic acid-targetingnucleic acid can comprise two nucleic acid molecules 1101/1110. Adouble-guide nucleic acid-targeting nucleic acid can comprise aplurality of non-native sequences 1115/1120/1125/1130. The non-nativesequence can be located at the 3′end, 5′ end or both 3′ and 5′ ends ofeach molecule of the nucleic acid-targeting nucleic acid. For example,the non-native sequence can be located at the 3′end, 5′ end or both 3′and 5′ ends of the first nucleic acid molecule 1105. The non-nativesequence can be located at the 3′end, 5′ end or both 3′ and 5′ ends ofthe second molecule 1110. A nucleic acid-targeting nucleic acid cancomprise a one or more non-native sequences in any of the enumeratedconfigurations in FIG. 11.

The disclosure provides for methods of use of tagged nucleicacid-targeting nucleic acids. The method can be performed using any ofthe site-directed polypeptides, nucleic acid-targeting nucleic acids,and complexes of site-directed polypeptides and nucleic acid-targetingnucleic acids as described herein. In some instances, a plurality oftagged nucleic acid-targeting nucleic acids can be contacted to aplurality of target nucleic acids. FIG. 12 depicts an exemplary methodof use for tagged nucleic acid-targeting nucleic acids. A tagged nucleicacid-targeting nucleic acid can comprise a spacer 1210 that canhybridize with a target nucleic acid 1205. The nucleic acid-targetingnucleic acid can comprise a non-native sequence (e.g., tag) 1220. Thenon-native sequence 1220 can be an RNA-binding protein binding sequence.In some instances, the non-native sequence 1220 can be a CRISPRRNA-binding protein binding sequence. The non-native sequence 1220 canbe bound by a RNA-binding protein 1215. The RNA-binding protein 1215 cancomprise a non-native sequence 1225 (e.g., a fusion, i.e., theRNA-binding protein 1215 can be a fusion polypeptide). The non-nativesequence (e.g., fusion) 1225 can alter the transcription of the targetnucleic acid and/or an exogenous nucleic acid. The non-native sequence(e.g., fusion) 1225 can comprise a first portion of a split system.

In some embodiments, a second nucleic acid-targeting nucleic acid,comprising a second spacer 1240 that can hybridize to a second targetnucleic acid 1245, can comprise a second non-native sequence (e.g., tag)1250. The second non-native sequence (e.g., tag) 1250 can be anRNA-binding protein binding sequence. The second non-native sequence(e.g., tag) 1250 can be a CRISPR RNA-binding protein binding sequence.The second non-native sequence 1250 can be bound by a RNA-bindingprotein 1235. The RNA-binding protein can comprise a non-native sequence1230 (e.g., fusion, i.e., the RNA-binding protein 1235 can be a fusion).The non-native sequence 1230 (e.g., fusion) can be a second portion of asplit system.

In some instances, the first portion of the split system 1225 and thesecond portion of the split system 1230 can be close together in space,such that the first portion of the split system 1225 and the secondportion of the split system 1230 interact 1255 to form an active splitsystem 1260. An active split system 1260 can refer to an unsplit system,wherein the first portion and the second portion form a whole piece ofthe split system. Activation of the split system can indicate that twotarget nucleic acids 1205/1245 are close together in space.

Methods

The disclosure provides for methods for contacting a target nucleic acidwith a complex comprising a site-directed polypeptide and a nucleicacid-targeting nucleic acid, and introducing one or more effectorproteins, wherein the one or more effector proteins comprises anon-native sequence and can bind to the modified nucleic acid-targetingnucleic acid. An effector protein can refer to any protein with afunctional effect. For example, an effector protein can compriseenzymatic activity, remodel biological molecules (e.g., foldingchaperones), be a scaffolding protein, and/or bind a small molecule ormetabolite. The effector protein can modify the target nucleic acid(e.g., cleavage, enzymatic modification, transcriptional modification).The methods of the disclosure provide for using the compositions of thedisclosure as biosensors. For example, the complexes (e.g., comprising amodified nucleic acid-targeting nucleic acid, a site-directedpolypeptide and/or an effector protein) can be used to monitor geneticmobility events, sense when sequences are close together inthree-dimensional space, and conditionally alter transcription.

Genetic Mobility Event

The disclosure provides for methods for determining the occurrence of agenetic mobility event. The method can be performed using any of thesite-directed polypeptides, nucleic acid-targeting nucleic acids, andcomplexes of site-directed polypeptides and nucleic acid-targetingnucleic acids as described herein. A genetic mobility event cancomprise, for example, a translocation, a recombination, an integration,a transposition, a horizontal gene transfer event, a transformation, atransduction, a conjugation, a gene conversion event, a duplication, atranslocation, an inversion, a deletion, a substitution, or anycombination thereof.

A genetic mobility event can comprise a recombination between genes. Therecombination can lead to deleterious gene products (e.g., the BCR-ABLrecombination which can contribute to breast cancer). Recombination caninclude, for example, homologous recombination, non-homologousrecombination (e.g., non-homologous end joining), and V(D)Jrecombination. Recombination can refer to chromosomal crossover.Recombination can occur during prophase I of meiosis (e.g., synapsis).Recombination can comprise double-stranded breakage of nucleic acidstrands of DNA, followed by formation of a holiday junction byrecombinases which can catalyze swapping of the DNA strands.

Genetic mobility events can cause disease. For example, chronicmyelogenous leukemia can result from a genetic mobility event.Translocation between chromosome 9 and 22 can result in a fusionBCR-Abl1 gene, which can result in the lengthening of one chromosome(e.g., 9), and the shortening of another chromosome (e.g., 22, i.e.,Philadelphia chromosome). The BCR-Abl1 translocation can lead to theproduction of a BCR-Abl fusion protein which can interact with receptors(e.g., interleukin-3 receptor) to promote cell division, leading tochronic myelogenous leukemia (CML). Other non-limiting exemplary geneticmobility events include BRD3-NUT, BRD4-NUT, KIAA1549-BRAF,FIG/GOPC-ROS1, ETV6-NTRK3, BCAS4-BCAS3, TBL1XR1-RGS17, ODZ4-NRG1,MALAT1-TFEB, APSCR1-TFE3, PRCC-TFE3, CLTC-TFE3, NONO-TFE3, SFPQ-TFE3,ETV6-NRTK3, EML4-ALK, EWSR1-ATF1, MN1-ETV6, CTNNB1-PLAG1, LIFR-PLAG1,TCEA1-PLAG1, FGFrl-PLAG1, CHCHD7-PLAG1, HMGA2-FHIT, HMGA-NFIB,CRTC1-MAM12, CRCT3-MAML2, EWSR1-POUF5F1, TMPRSS1-ERG, TMPRSS2-ETV4,TMPRSS2-ETV5, HNRNPA2B1-ETV1, HERV-K-ETV1, C150RF21-ETV1, SLC45A3-ETV1,SLC45A3-ETV5, SLC45A3-ELK4, KLK2-ETV4, CANT1-ETV4, RET-PTC1/CCDC6,RET-PTC2/PRKAR1A, RET-PTC3,4/NCOA4, RET-PTC5/GOLGA5, RET-PTC6/TRIM24,RET-PTC7/TRIM33, RET-PTC8/KTN1, RET-PTC9/RFG9, RET-PTCM1, TFG-NTRK1,TPM3-NRTK1, TPR-NRTK1, RET-D10S170, ELKS-RET, HOOKS3-RET, RFP-RET,AKAP9-BRAF, and PAX8-PPARG.

Diseases that can be caused by genetic mobility events can includeCharcot-Marie-Tooth disease type 1A (CMT1A), juvenile nephronophtisis(NPH), X-linked icthyosis, familial growth hormone deficiency type 1A,fascioscapulohumeral muscular dystrophy (FSHD), α-thalassemia,hemophilia A, Hunter syndrome (i.e., mucopolysaccharidosis II),Emery-Dreifuss musclar dystrophy, Hemoglobin Lepore, steroid21-hydroxylase deficiency, glucocorticoid-suppressiblehyperaldosteronism (GSH), color-blindness (e.g., visual dichromacy),autosomal recessive spinal muscular atrophy (SMA), cancer, T-cell acutelymphoblastic leukemia (T-ALL), aggressive midline carcinoma,Astrocytoma, Secretory breast carcinoma, Breast cancer, Kidneycarcinoma, Mesoblastic nephroma, Lung adenocarcinoma, Melanoma,Meningioma, pleomorphic adenoma, mucoepidermoid cancer, Prostatecarcinoma, Thyroid carcinoma, and acute promyelocytic leukemia.

The methods of the disclosure provide for determining the occurrence ofa genetic mobility event in which a target nucleic acid can be contactedwith two complexes, each complex comprising a site-directed polypeptideand a modified nucleic acid-targeting nucleic acid, and two or moreeffector proteins can be introduced, wherein the two or more effectorproteins can bind to the modified nucleic acid-targeting nucleic acids,wherein one of the two or more effector proteins comprises a non-nativesequence that is a first piece of a split system and one of the two ormore effector proteins comprises a non-native sequence that is a secondpiece of the split system. A split system can refer to a protein complexcomposed of two or more protein fragments that individually are notfluorescent, but, when formed into a complex, result in a functional(that is, fluorescing) fluorescent protein complex. Individual proteinfragments of a split system (e.g., split fluorescent protein) can bereferred to as “complementing fragments” or “complementary fragments”.Complementing fragments which can spontaneously assemble into afunctional fluorescent protein complex are known as self-complementing,self-assembling, or spontaneously-associating complementing fragments.For example, a split system can comprise GFP. In a GFP split system,complementary fragments are derived from the three dimensional structureof GFP, which includes eleven anti-parallel outer beta strands and oneinner alpha strand. A first fragment can comprise one of the elevenbeta-strands of the GFP molecule (e.g., GFP S11), and a second fragmentcan comprise the remaining strands (e.g., GFP S1-10).

Prior to the genetic mobility event the target nucleic acid sequencetargetable by one complex can be far apart from the target nucleic acidsequence targetable by another sequence. The distance between the twotarget nucleic acid sequences can comprise at least about 0.1, 0.5, 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more Kb. The distance between the twotarget nucleic acid sequences can comprise at most about 0.1, 0.5, 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more Kb. The two target nucleic acidsequences can be located on different chromosomes. The two targetnucleic acid sequences can be located on the same chromosome.

Prior to the genetic mobility event the effector proteins that comprisepieces of the split system may not be able to interact with each other(e.g., the split system can be inactive). After the genetic mobilityevent, the target nucleic acid sequence targetable by one complex may belocated in close proximity to the target nucleic acid sequencetargetable by the other complex. After the genetic mobility event, theeffector proteins that comprise pieces of the split system may be ableto interact with each other, thereby activating the split system.

The activated split system can indicate the occurrence of the geneticmobility event. For example, if the activated split system is afluorescent protein split system, then prior to the genetic mobilityevent fluorescence may not be detected in the sample. In some instances,the levels of fluorescence of the inactive split system (e.g.,background levels) may be 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5or more fold less fluorescent compared to a control sample (e.g., cell)that does not comprise the split system. In some instances, the levelsof fluorescence of the inactive split system (e.g., background level)may be 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or more fold morefluorescent than a control sample (e.g., cell) that does not comprisethe split system.

After the genetic mobility event, the two split pieces can unite to forman active fluorescent protein, and fluorescence can be detected in thesample. An active split system can result in at least about a 0.1, 0.5,1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more fold increase in fluorescence. Anactive split system can result in at most about a 0.1, 0.5, 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more fold increase in fluorescence.

Detection of a genetic mobility can be used to genotype a subject (e.g.,a patient). A genotype can be indicative of a disease. The detection ofa genetic mobility event can be used to diagnose a subject. The geneticand diagnostic information obtained from the methods described hereincan be communicated to a subject. The genetic and diagnostic informationobtained from the methods described herein can be used to develop asubject-specific treatment plan. For example, if the data obtained fromthe methods of the disclosure indicate that a patient has a genotypethat makes them resistant to a particular therapeutic regimen, a newtreatment plan can be made for the subject.

Altering Transcription

The methods of the disclosure can provide for altering the transcriptionof a nucleic acid. The method can be performed using any of thesite-directed polypeptides, nucleic acid-targeting nucleic acids, andcomplexes of site-directed polypeptides and nucleic acid-targetingnucleic acids as described herein. The method provides for contacting atarget nucleic acid with two complexes, each complex comprising asite-directed polypeptide and a modified nucleic acid-targeting nucleicacid, and introducing two or more effector proteins, wherein the two ormore effector proteins can bind to the modified nucleic acid-targetingnucleic acids, wherein the one of the two or more effector proteinscomprises a non-native sequence that is a first piece of a splittranscription factor system and one of the two or more effector proteinscomprises a non-native sequence that is a second piece of the splittranscription factor system, and wherein an interaction between thefirst piece and the second piece of the split system forms atranscription factor that alters transcription of the nucleic acid.

The transcription factor can alter transcription levels of a nucleicacid and/or a target nucleic acid. Altered transcription can includeincreased transcription levels and/or decreased transcription levels. Atranscription factor can alter transcription levels more than 2-fold,3-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold or more higher orlower than unaltered transcription levels. A transcription factor canalter transcription levels less than 2-fold, 3-fold, 5-fold, 10-fold,50-fold, 100-fold, 1000-fold or more higher or lower than unalteredtranscription levels.

The transcription factor can alter the transcription of a target nucleicacid and/or an exogenous nucleic acid. A target nucleic acid can be thenucleic acid that is contacted by the complex comprising thesite-directed polypeptide and the nucleic acid-targeting nucleic acid.An exogenous nucleic acid can comprise a donor polynucleotide, aplasmid, and/or a target nucleic acid.

An exogenous nucleic acid can comprise a polynucleotide encoding genesinvolved in apoptosis. Suitable genes involved in apoptosis can includetumor necrosis factor (TNF), TNF-R1, TNF-R2, TNF receptor-associateddeath domain (TRADD), Fas receptor and Fas ligand, caspases (e.g.,caspase-3, caspase-8, caspase-10), APAF-1, FADD, and apoptosis inducingfactor (AIF). An exogenous nucleic acid can comprise a polynucleotideencoding genes that result in cell lysis. Suitable genes can include theAdenovirus death protein (ADP), defensins, membrane-permeabilizing lyticpeptides derived from c-FLIP, procaspases, cell-penetrating peptidese.g. HIV TAT. An exogenous nucleic acid can comprise a polynucleotideencoding an antigen that can result in recruitment of immune cells tothe cell location (e.g., MHC class peptides). An exogenous nucleic acidcan comprise a polynucleotide encoding a nucleic-acid targeting nucleicacid that targets sequences that occur many times within the genome(e.g., microsatellites, tandem repeats), resulting in large scale genomefragmentation and cell-death.

Modification of Target Nucleic Acid

The disclosure provides for methods to modify a target nucleic acidusing the nucleic acid-targeting nucleic acid of the disclosure. Themethod can be performed using any of the site-directed polypeptides,nucleic acid-targeting nucleic acids, and complexes of site-directedpolypeptides and nucleic acid-targeting nucleic acids as describedherein. For example, a target nucleic acid can be contacted with acomplex comprising a site-directed polypeptide, a nucleic acid-targetingnucleic acid, and one or more effector proteins, wherein the one or moreeffector proteins comprises a non-native sequence and can bind to themodified nucleic acid-targeting nucleic acid. The non-native sequencecan confer an enzymatic activity and/or transcriptional activity of theeffector protein can modify the target nucleic acid. For example, if theeffector protein comprises a non-native sequence corresponding to amethyltransferase, then the methyltransferase may be able to methylatethe target nucleic acid. The modification of the target nucleic acid mayoccur at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100 or more nucleotides away from the either the 5′ or 3′ end ofthe target nucleic acid. The modification of the target nucleic acid mayoccur at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80,90, 100 or more nucleotides away from the either the 5′ or 3′ end of thetarget nucleic acid. The modification can occur on a separate nucleicacid that does not comprise the target nucleic acid (e.g., anotherchromosome).

Exemplary modifications can comprise methylation, demethylation,acetylation, deacetylation, ubiquitination, deubiquitination,deamination, alkylation, depurination, oxidation, pyrimidine dimerformation, transposition, recombination, chain elongation, ligation,glycosylation. Phosphorylation, dephosphorylation, adenylation,deadenylation, SUMOylation, deSUMOylation, ribosylation, deribosylation,myristoylation, remodelling, cleavage, oxidoreduction, hydrolation, andisomerization.

Determining a Genotype and Treatment

The disclosure provides for methods for treating a disease using thenucleic acid-targeting nucleic acid of the disclosure. The method can beperformed using any of the site-directed polypeptides, nucleicacid-targeting nucleic acids, and complexes of site-directedpolypeptides and nucleic acid-targeting nucleic acids as describedherein. For example, using the split system described herein, thepresence of two or more target nucleic acids close together in space(e.g., in a genetic mobility event, in chromatin structure, or on alinear nucleic acid) can be indicative of a genotype (e.g, of asubject). A genotype can refer to the presence or absence of aparticular sequence of nucleic acid, a nucleotide polymorphism (i.e.,either a single nucleotide polymorphism, or a multi-nucleotidepolymorphism), an allelic variant, or any other indication of thesequence of a nucleic acid. The genotype can indicate whether a patientsuffers from a disease and/or is predisposed to contract a disease.

Determining a genotype can include, for example, determining if asubject comprises a mutant sequence (e.g., nucleic acid sequencecomprising a mutation). In some instances, a first nucleicacid-targeting nucleic acid comprising the appropriate components asdescribed herein to comprise a first part of a split system can bedesigned to target a region near a predicted mutant sequence. In someinstances, a second nucleic acid-targeting nucleic acid comprising theappropriate components as described herein to comprise a second part ofthe split system can be designed to target a region comprising thepredicted mutant sequence. If the mutant sequence does exist, the secondnucleic acid-targeting nucleic acid can bind to it, and the two parts ofthe split system can interact. The interaction can generate a signalwhich can be indicative of the presence of a mutant sequence.

A genotype can be identified by a biomarker. A biomarker can beindicative of any physiological process. A biomarker can serve as aindicator of efficacy of a treatment (e.g., drug treatment). A biomarkercan be a nucleic acid, a polypeptide, an analyte, a solute, a smallmolecule, an ion, an atom, a modification to a nucleic acid and/orpolypeptide, and/or a degradation product. A biomarker can refer torelative expression levels of a nucleic acid and/or a polypeptide.

A subject-specific treatment plan may be identified from determining thegenotype of the subject using the methods of the disclosure. Forexample, if a subject comprises a certain genotype known to beunresponsive to a particular therapy, then the subject can be treatedwith a different therapy. Determining of genotype can allow a subject tobe selected or deselected for a clinical trial.

Determination of the genotype can be communicated from a caregiver to asubject (e.g., from a doctor to a patient, or from a person performingthe genotype analysis to a customer). The communication can occur inperson (e.g., in a doctor's office), over the phone, in writing, orelectronically. The communication can further inform the subject of asubject-specific treatment regimen determined from the genotype of thesubject.

The method can be performed more than once (e.g., iteratively) in asubject. For example, the genotype of a subject can be determined, acourse of treatment can be prescribed for the subject, the genotype ofthe subject can be determined again. The two genotypes can be comparedto determine the effectiveness of the course of treatment. The treatmentplan can be altered based on the comparison of the genotypes.

Location of Sequences in Three-Dimensional Space

In some instances, the disclosure provide for a method for determiningthe location of sequences in three-dimensional space in a cell. Themethod can be performed using any of the site-directed polypeptides,nucleic acid-targeting nucleic acids, and complexes of site-directedpolypeptides and nucleic acid-targeting nucleic acids as describedherein. Determining the three-dimensional organization of chromatin andnucleic acid can be important for understanding gene regulation such astranscriptional activation and/or repression of genes. In someinstances, the method comprises contacting a target nucleic acid withtwo complexes, wherein each complex binds to its cognate target nucleicacid. The complexes can comprise a site-directed polypeptide and anucleic acid-target nucleic acid of the disclosure. Two or more effectorproteins can be introduced, wherein the each of the two or more effectorproteins binds to a complex. The effector proteins can be similar to thesplit system described above, wherein each effector protein can comprisean inactive fragment of a whole polypeptide. When the effector proteinsare far apart in space, the effector proteins are inactive (e.g., nosignal is detected). When the effector proteins are close enough inspace to interact, they can form a detectable active polypeptide.

The effector proteins can be part of a split affinity tag system. In asplit affinity tag system, the two inactive polypeptide fragments of thesystem can correspond to two inactive fragments of an affinity tag. Whenthe two fragments bind together, the whole affinity tag is restored,such that the affinity tag can be detectable by a binding agent. Abinding agent can refer to a molecule that can bind and purify theaffinity tag. Examples of binding agents can include antibodies,antibody-conjugated beads, and small-molecule conjugated beads.

Introduction of the complexes and polypeptides of the disclosure canoccur by viral or bacteriophage infection, transfection, conjugation,protoplast fusion, lipofection, electroporation, calcium phosphateprecipitation, polyethyleneimine (PEI)-mediated transfection,DEAE-dextran mediated transfection, liposome-mediated transfection,particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticlemediated nucleic acid delivery, and the like.

The cells can be cultured with the complexes for at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more days. The cells can be cultured with thecomplexes for at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. Afteran appropriate period of time (e.g, a period of time to allow thecomplexes to bind to their target nucleic acid), the cells can be lysed.

The cells can be crosslinked before they are lysed. Fixed orcross-linking cells can stabilize protein-DNA complexes in the cell.Suitable fixatives and cross-linkers can include, formaldehyde,glutaraldehyde, ethanol-based fixatives, methanol-based fixatives,acetone, acetic acid, osmium tetraoxide, potassium dichromate, chromicacid, potassium permanganate, mercurials, picrates, formalin,paraformaldehyde, amine-reactive NHS-ester crosslinkers such asbis[sulfosuccinimidyl] suberate (BS3),3,3′-dithiobis[sulfosuccinimidylpropionate] (DTSSP), ethylene glycolbis[sulfosuccinimidylsuccinate (sulfo-EGS), disuccinimidyl glutarate(DSG), dithiobis[succinimidyl propionate] (DSP), disuccinimidyl suberate(DSS), ethylene glycol bis[succinimidylsuccinate] (EGS),NHS-ester/diazirine crosslinkers such as NHS-diazirine,NHS-LC-diazirine, NHS-SS-diazirine, sulfo-NHS-diazirine,sulfo-NHS-LC-diazirine, and sulfo-NHS-SS-diazirine.

Lysed cells can be contacted with a binding agent (e.g, an antibody)that is directed to bind to the affinity tag. The contacting can occurin a test-tube. The contacting can occur in a chromatographic setting(e.g., an affinity chromatography column). Contacting with the bindingagent can occur for at least 1 minute, 5 minutes, 10 minutes, 15minutes, 20 minutes, 25 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours,20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 or more hours.Contacting with the binding agent can occur for at most 1 minute, 5minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40hours, 45 or more hours. In some instances, contacting with a bindingagent occurs prior to cell lysis.

The complexes can be purified with the binding agent. The purifiedcomplexes can be subjected to nucleic acid purification techniques toseparate the target nucleic acid from the complexes. Nucleic acidpurification techniques can include spin column separation,precipitation, and electrophoresis.

The nucleic acid (e.g., nucleic acid comprising the target nucleic acid)can be subjected to sequencing methodologies. The nucleic acid can beprepared for sequencing analysis by ligation of one or more adaptors.Sequenced nucleic acids can be analyzed to identify polymorphisms,diagnose a disease, determine a course of treatment for a disease,and/or determine the three-dimensional structure of the genome.

Tagged Nucleic Acid-Targeting Nucleic Acids with Linkers

The disclosure provides for compositions and methods for generating andusing tagged nucleic acid-targeting nucleic acids. FIG. 13A depicts anexemplary untagged nucleic acid-targeting nucleic acid. An untaggednucleic acid-targeting nucleic acid can comprise a protospacer (PS), aminimum CRISPR repeat (MCR), a single guide connector (SGC), a minimumtracrRNA sequence (MtS), a 3′ tracrRNA sequence (3TS), and tracrRNAextension (TE). A tagged nucleic acid-targeting nucleic acid comprisinga linker can refer to any of the nucleic acid-target nucleic acidsdescribed herein, and comprising a linker at either the 5′ end, the 3′end, or both the 5′ and 3′ end of the nucleic acid-targeting nucleicacid.

A nucleic acid-targeting nucleic acid can comprise a non-native sequenceas depicted in FIG. 13B. The non-native sequence can be referred to as atag. The tag can be fused to either the 5′ end, the 3′ end or both the5′ and 3′ end of the nucleic acid-targeting nucleic acid. The non-nativesequence can comprise a binding sequence for an RNA-binding protein. TheRNA-binding protein can be Csy4. The non-native sequence can be fused tothe protospacer sequence of the nucleic acid-targeting nucleic acid.

The non-native fusion can be separated from the nucleic acid-targetingnucleic acid by a linker. FIG. 14 depicts an exemplary linker (e.g., Taglinker), separating the non-native sequence (e.g, Csy4 hairpin) from theprotospacer of the nucleic acid-targeting nucleic acid. The linkersequence can be complementary to the target nucleic acid. The linkersequence can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moremismatches with the target nucleic acid. The linker sequence cancomprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mismatches withthe target nucleic acid. In some instances, the fewer mismatches betweenthe linker and target nucleic acid, the better cleavage efficiency ofthe Cas9:nucleic acid-targeting nucleic acid complex.

The linker sequence can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60 ormore nucleotides in length. The linker sequence can be at most 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, 55, 60 or more nucleotides in length.

Multiplexed Genetic Targeting Agents

General Overview

This disclosure describes methods, compositions, systems, and/or kitsfor multiplexed genome engineering. In some embodiments of thedisclosure a site-directed polypeptide can comprise a nucleicacid-targeting nucleic acid, thereby forming a complex. The complex canbe contacted with a target nucleic acid. The target nucleic acid can becleaved, and/or modified by the complex. The methods, compositions,systems, and/or kits of the disclosure can be useful in modifyingmultiple target nucleic acids quickly, efficiently, and/orsimultaneously. The method can be performed using any of thesite-directed polypeptides, nucleic acid-targeting nucleic acids, andcomplexes of site-directed polypeptides and nucleic acid-targetingnucleic acids as described herein.

FIG. 15 depicts an exemplary embodiment of the methods of thedisclosure. A nucleic acid (e.g., a nucleic acid-targeting nucleic acid)1505 can be fused to a non-native sequence (e.g., a moiety, anendoribonuclease binding sequence, ribozyme) 1510, thereby forming anucleic acid module 1512. The nucleic acid module 1512 (e.g., comprisingthe nucleic acid fused to a non-native sequence) can be conjugated intandem, thereby forming a multiplexed genetic targeting agent (e.g.,polymodule, e.g., array) 1511. The multiplexed genetic targeting agent1511 can comprise RNA. The multiplexed genetic targeting agent can becontacted 1515 with one or more endoribonucleases 1520. Theendoribonucleases can bind to the non-native sequence 1510. The boundendoribonuclease can cleave a nucleic acid module 1512 of themultiplexed genetic targeting agent 1511 at a prescribed locationdefined by the non-native sequence 1510. The cleavage 1525 can process(e.g., liberate) individual nucleic acid modules 1512. In someembodiments, the processed nucleic acid modules 1512 can comprise all,some, or none, of the non-native sequence 1510. The processed nucleicacid modules 1512 can be bound by a site-directed polypeptide 1530,thereby forming a complex 1531. The complex 1531 can be targeted 1535 toa target nucleic acid 1540. The target nucleic acid 1540 can by cleavedand/or modified by the complex 1531.

Multiplexed Genetic Targeting Agents

A multiplexed genetic targeting agent can be used in modifying multipletarget nucleic acids at the same time, and/or in stoichiometric amounts.A multiplexed genetic targeting agent can be any nucleic acid-targetingnucleic acid as described herein in tandem. A multiplexed genetictargeting agent can refer to a continuous nucleic acid moleculecomprising one or more nucleic acid modules. A nucleic acid module cancomprise a nucleic acid and a non-native sequence (e.g., a moiety,endoribonuclease binding sequence, ribozyme). The nucleic acid can benon-coding RNA such as microRNA (miRNA), short interfering RNA (siRNA),long non-coding RNA (lncRNA, or lincRNA), endogenous siRNA (endo-siRNA),piwi-interacting RNA (piRNA), trans-acting short interfering RNA(tasiRNA), repeat-associated small interfering RNA (rasiRNA), smallnucleolar RNA (snoRNA), small nuclear RNA (snRNA), transfer RNA (tRNA),and ribosomal RNA (rRNA), or any combination thereof. The nucleic acidcan be a coding RNA (e.g., a mRNA). The nucleic acid can be any type ofRNA. In some embodiments, the nucleic acid can be a nucleicacid-targeting nucleic acid.

The non-native sequence can be located at the 3′ end of the nucleic acidmodule. The non-native sequence can be located at the 5′ end of thenucleic acid module. The non-native sequence can be located at both the3′ end and the 5′ end of the nucleic acid module. The non-nativesequence can comprise a sequence that can bind to a endoribonuclease(e.g., endoribonuclease binding sequence). The non-native sequence canbe a sequence that is sequence-specifically recognized by anendoribonuclease (e.g., RNase T1 cleaves unpaired G bases, RNase T2cleaves 3′end of As, RNase U2 cleaves 3′end of unpaired A bases). Thenon-native sequence can be a sequence that is structurally recognized byan endoribonuclease (e.g., hairpin structure, single-stranded-doublestranded junctions, e.g., Drosha recognizes a single-stranded-doublestranded junction within a hairpin). The non-native sequence cancomprise a sequence that can bind to a CRISPR system endoribonuclease(e.g., Csy4, Cas5, and/or Cas6 protein). The non-native sequence cancomprise a nucleotide sequence having at least or at most about about40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, nucleotidesequence identity and/or sequence similarity to one of the followingsequences:

5′-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3′;5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′.5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′,5′-GUCGCCCCCCACGCGGGGGCGUGGAUUGAAAC-3′;5′-CCAGCCGCCUUCGGGCGGCUGUGUGUUGAAAC-3′;5′-GUCGCACUCUACAUGAGUGCGUGGAUUGAAAU-3′;5′-UGUCGCACCUUAUAUAGGUGCGUGGAUUGAAAU-3′; and5′-GUCGCGCCCCGCAUGGGGCGCGUGGAUUGAAA-3′.

In some embodiments, wherein the non-native sequence comprises anendoribonuclease binding sequence, the nucleic acid modules can be boundby the same endoribonuclease. The nucleic acid modules may not comprisethe same endoribonuclease binding sequence. The nucleic acid modules maycomprise different endoribonuclease binding sequences. The differentendoribonuclease binding sequences can be bound by the sameendoribonuclease. In some embodiments, the nucleic acid modules can bebound by different endoribonucleases.

The moiety can comprise a ribozyme. The ribozyme can cleave itself,thereby liberating each module of the multiplexed genetic targetingagent. Suitable ribozymes can include peptidyl transferase 23S rRNA,RnaseP, Group I introns, Group II introns, GIR1 branching ribozyme,Leadzyme, hairpin ribozymes, hammerhead ribozymes, HDV ribozymes, CPEB3ribozymes, VS ribozymes, glmS ribozyme, CoTC ribozyme, an syntheticribozymes.

The nucleic acids of the nucleic acid modules of the multiplexed genetictargeting agent can be identical. The nucleic acid modules can differ by1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or morenucleotides. For example, different nucleic acid modules can differ inthe spacer region of the nucleic acid module, thereby targeting thenucleic acid module to a different target nucleic acid. In someinstances, different nucleic acid modules can differ in the spacerregion of the nucleic acid module, yet still target the same targetnucleic acid. The nucleic acid modules can target the same targetnucleic acid. The nucleic acid modules can target one or more targetnucleic acids.

A nucleic acid module can comprise a regulatory sequence that can allowfor appropriate translation or amplification of the nucleic acid module.For example, an nucleic acid module can comprise a promoter, a TATA box,an enhancer element, a transcription termination element, aribosome-binding site, a 3′ un-translated region, a 5′ un-translatedregion, a 5′ cap sequence, a 3′ poly adenylation sequence, an RNAstability element, and the like.

Methods

The disclosure provides for methods for the modification of multipletarget nucleic acids, simulataneously, through the use of a multiplexedgenetic targeting agent. A site-directed polypeptide, anendoribonuclease, and a multiplexed genetic targeting agent can beintroduced into a host cell. A vector of the disclosure (e.g.,comprising a multiplexed genetic targeting agent, an endoribonucleaseand/or a site-directed polypeptide) can be introduced into a host cell.In some instances, more than one endoribonuclease and/or multiplexedgenetic targeting agent can be introduced into cells. If a multiplexedgenetic targeting agent comprises different types of moieties, where themoieties are different endoribonuclease binding sequences, then one ormore endoribonucleases corresponding to the types of binding sequencesin the multiplexed genetic targeting agent may be introduced into cells.

Introduction can occur by any means to introduce a nucleic acid into acell such viral or bacteriophage infection, transfection, conjugation,protoplast fusion, lipofection, electroporation, calcium phosphateprecipitation, polyethyleneimine (PEI)-mediated transfection,DEAE-dextran mediated transfection, liposome-mediated transfection,particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.The vector can be transiently expressed in the host cell. The vector canbe stably expressed in the host cell (e.g., by stably integrating intothe genome of the host cell).

In instances where a moiety comprises an endoribonuclease bindingsequence, an endoribonuclease can be expressed and can bind to theendoribonuclease binding site on the multiplexed genetic targetingagent. The endoribonuclease can cleave the multiplexed genetic targetingagent into individual nucleic acid modules.

In instances where a moiety comprises a ribozyme, an endoribonucleasemay not be required to be expressed in a host cell. The ribozyme cancleave itself, thereby resulting in cleavage of the multiplexed genetictargeting agent into individual nucleic acid modules.

Individual (e.g., cleaved) nucleic acid modules can comprise all, some,or none, of the moiety (e.g., endoribonuclease binding sequence). Forexample, the liberated (e.g., processed) nucleic acid module can besubjected to exonuclease trimming and/or degradation that may result inremoval of the 5′ and/or 3′ end of the nucleic acid module. In suchinstances, exonuclease trimming and/or degradation may result in theremoval of all, part, or none of the moiety (e.g., endoribonucleasebinding sequence).

The liberated (e.g., processed) nucleic acid module can bind to asite-directed polypeptide thereby forming a complex. The complex can beguided to a target nucleic acid by the nucleic acid-targeting nucleicacid which can hybridize with the target nucleic acid in asequence-specific manner. Once hybridized, the site-directed polypeptideof the complex can modify the target nucleic acid (e.g., cleave thetarget nucleic acid). In some instances, the modification comprisesintroduction of a double-stranded break in the target nucleic acid. Insome instances, the modification comprises introduction of asingle-stranded break in the target nucleic acid.

In some embodiments, one or more donor polynucleotides and/or vectorsencoding the same can introduced into the cell. One or more donorpolynucleotides can be incorporated into the modified (e.g., cleaved)target nucleic acids, thereby resulting in an insertion. The same donorpolynucleotide can be incorporated into multiple cleavage sites oftarget nucleic acids. One or more donor polynucleotides can beincorporated into one or more cleavage sites of target nucleic acids.This can be referred to as multiplex genome engineering. In someinstances, no donor polynucleotide and/or vector encoding the same maybe introduced into the cells. In these instances, the modified targetnucleic acid can comprise a deletion.

Stoichiometric Delivery of Nucleic Acids

General Overview

The disclosure provides for compositions, methods, and kits forstoichiometric delivery of a nucleic acid to a cell and/or subcellularlocalization. The stoichiometric delivery may be mediated by a complex.FIG. 16 depicts an exemplary complex for stoichiometric delivery of aplurality of nucleic acids to a cell and/or subcellular location. Thecomplex can comprise a plurality of nucleic acids 1605. Each nucleicacid can comprise a nucleic acid-binding protein binding site 1610. Thenucleic acid-binding protein binding sites 1610 can all be the samesequences, different sequences, or some can be same sequences and somecan be different sequences. In some embodiments, the nucleicacid-binding protein binding sites can bind a Cas6, Cas5, or Csy4 familymember. The complex can comprise a tandem fusion polypeptide 1630. Thetandem fusion polypeptide can comprise nucleic acid-binding proteins1625 fused together in tandem. The nucleic acid-binding proteins can beseparated by a linker 1620. The nucleic acid-binding proteins 1625 canbe the same protein, can be different proteins, or some can be the sameproteins and some can be different proteins. The nucleic acid-bindingproteins 1625 can be Csy4 proteins. The nucleic acid-binding proteins1625 can bind the nucleic acid-binding protein binding site 1610 on thenucleic acid 1605. The tandem fusion polypeptide 1630 can comprise anon-native sequence 1615. In some instances, the non-native sequence isa subcellular (e.g., nuclear) localization sequence. In someembodiments, the nucleic acid 1605 can encode a non-native sequence(e.g. a subcellular, (e.g., nuclear) localization sequence). The complexcan be introduced 1635 into cells, wherein one or more of the nucleicacids 105 can be translated into polypeptides 1640. A translatedpolypeptide 1640 can bind and cleave the nucleic acid-binding proteinbinding site 1610 on the nucleic acid 1605. The cleavage 1645 canliberate the nucleic acid 1650 which can be a nucleic acid-targetingnucleic acid. The liberated nucleic acid 1650 can bind to a translatedpolypeptide 1645 (e.g., a site-directed polypeptide), thereby forming aunit. The translated polypeptide 1645 can comprise a nuclearlocalization signal. The unit can translocate to the nucleus, whereinthe unit can be guided to a target nucleic acid hybridizable with thespacer of the liberated nucleic acid 1650. The unit can be hybridized toa target nucleic acid. The site-directed polypeptide of the unit cancleave the target nucleic acid. The cleavage of the target nucleic acidcan be referred to as genome engineering. The method can be performedusing any of the site-directed polypeptides, nucleic acid-targetingnucleic acids, and complexes of site-directed polypeptides and nucleicacid-targeting nucleic acids as described herein.

In some embodiments, multiple nucleic acid-targeting nucleic acids canbe stoichiometrically delivered to a cell and/or subcellular location.FIG. 17 depicts an exemplary complex for stoichiometric delivery of aplurality of nucleic acids. The complex can comprise a plurality ofnucleic acids 1705. Each nucleic acid can comprise a plurality ofnucleic acid-binding protein binding sites 1710/1711. The nucleicacid-binding protein binding sites 1710/1711 can all be the samesequences, different sequences, or some can be same sequences and somecan be different sequences. In some embodiments, the nucleicacid-binding protein binding sites 1710/1711 can bind a Cas6, Cas5, orCsy4 family member. The complex can comprise a tandem fusion polypeptide1730. The tandem fusion polypeptide can comprise nucleic acid-bindingproteins 1725 fused together in tandem. The nucleic acid-bindingproteins can be separated by a linker 1720. The nucleic acid-bindingproteins 1725 can be the same protein, can be different proteins, orsome can be the same proteins and some can be different proteins. TheRNA-binding proteins 1725 can be a combination of Csy4, Cas5, and Cas6polypeptides. The nucleic acid-binding proteins 1725 can bind thenucleic acid-binding protein binding site 1710 on the nucleic acid 1705.The tandem fusion polypeptide 1730 can comprise a non-native sequence1715. In some instances, the non-native sequence is a subcellular (e.g.,nuclear) localization sequence. In some embodiments, the nucleic acid1705 can encode for a non-native sequence (e.g. a subcellular, (e.g.,nuclear) localization sequence). The complex can be introduced intocells 1735, wherein one or more of the nucleic acids can be translatedinto polypeptides 1740/1750. A translated polypeptide 1740 can bind andcleave the nucleic acid-binding protein binding site 1711 on the nucleicacid 1705. The cleavage 1745 can liberate the nucleic acid 1755, whichcan be a nucleic acid-targeting nucleic acid and/or a donorpolynucleotide. The liberated nucleic acid 1755 can bind to a translatedpolypeptide 1750 (e.g., a site-directed polypeptide), thereby forming aunit. In some instances, the translated polypeptide 1750 comprises anuclear localization signal. The unit can translocate to the nucleus,wherein the unit can be guided to a target nucleic acid hybridizablewith the spacer of the liberated RNA 1755. The unit can be hybridized toa target nucleic acid. The site-directed polypeptide of the unit cancleave the target nucleic acid.

Methods

The disclosure provides for methods for stoichiometric delivery ofnucleic acids to a cell (e.g., stoichiometrically deliverable nucleicacids). The method can comprise binding a tandem fusion polypeptide to aplurality of stoichiometrically deliverable nucleic acids, therebyforming a complex. The complex can comprise stoichiometric amounts ofthe nucleic acids (e.g., the complex can comprise the plurality ofnucleic acids in a prescribed ratio and/or amount). 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more nucleic acids can be stoichiometrically delivered. Insome instances, 3 stoichiometrically deliverable nucleic acids can bestoichiometrically delivered. In some instances, 4 stoichiometricallydeliverable nucleic acids can be stoichiometrically delivered.

The stoichiometrically deliverable nucleic acid can encode for apolypeptide or a non-coding RNA. The polypeptide may be a CRISPR systempolypeptide (e.g., a site-directed polypeptide, an endoribonuclease).The stoichiometrically deliverable nucleic acid can encode for more thanone polypeptide. The stoichiometrically deliverable nucleic acid cancomprise a plurality of stoichiometrically deliverable nucleic acids(e.g., in an array). The stoichiometrically deliverable nucleic acid canencode for a non-coding RNA. Examples of non-coding RNAs can includemicroRNA (miRNA), short interfering RNA (siRNA), long non-coding RNA(lncRNA, or lincRNA), endogenous siRNA (endo-siRNA), piwi-interactingRNA (piRNA), trans-acting short interfering RNA (tasiRNA),repeat-associated small interfering RNA (rasiRNA), small nucleolar RNA(snoRNA), small nuclear RNA (snRNA), transfer RNA (tRNA), and ribosomalRNA (rRNA). The stoichiometrically deliverable nucleic acid can be RNA.

The stoichiometrically deliverable nucleic acid can encode for anon-native sequence. In some instances, the stoichiometricallydeliverable nucleic acid encodes for a non-native sequence such thatwhen a polypeptide is translated from a stoichiometrically deliverablenucleic acid encoding a polypeptide, the polypeptide is fused to thenon-native sequence (e.g., thereby generating a fusion protein). Thenon-native sequence can be a peptide affinity tag. The non-nativesequence (e.g., peptide affinity tag) can be located at the N-terminusof the polypeptide, the C-terminus of the polypeptide, or any locationwithin the polypeptide (e.g., a surface accessible loop). In someembodiments, the non-native sequence is a nuclear localization signal(NLS). A NLS can be monopartite or bipartite sequence. The NLS can berecognized by nuclear import machinery (e.g., importins). A NLS can be asmall peptide (e.g., PKKKRKV of the SV40 large t-antigen). A NLS can bea polypeptide domain (e.g., acidic M9 domain of hnRNP A1).

The non-native sequence can be a nucleic acid affinity tag (e.g.,nucleic acid localization signal). For example, a stoichiometricallydeliverable nucleic acid encoding a DNA (e.g., a donor polynucleotide)can comprise a nucleic acid localization signal which can localize theDNA to the nucleus. Such nucleic acid localization signals can include,for example, peptide-nucleic acid (PNA) sequences.

The stoichiometrically deliverable nucleic acids can comprise regulatorysequences that can allow for appropriate translation or amplification ofthe nucleic acid. For example, an nucleic acid can comprise a promoter,a TATA box, an enhancer element, a transcription termination element, aDNA stability element, a ribosome-binding site, a 3′ un-translatedregion, a 5′ un-translated region, a 5′ cap sequence, a 3′ polyadenylation sequence, an RNA stability element, and the like.

The nucleic acid can comprise a nucleic acid-binding protein bindingsite. The nucleic acid-binding protein binding site can be bound by annucleic acid-binding protein. The nucleic acid-binding protein bindingsite can be bound by a CRISPR polypeptide (e.g., a site-directedpolypeptide, an endoribonuclease). The nucleic acid-binding proteinbinding site can be bound by a Cas5 or Cas6 family polypeptide. Thenucleic acid-binding protein binding site can be bound by a Csy4, Cas5,or Cas6 polypeptide. Some examples of nucleic acid-binding proteinbinding sites can include, for example, sequences that can be bound byRNA-binding proteins such as the MS2 binding sequence, the U1A bindingsequence, the boxB sequence, the eIF4A sequence, hairpins, sequencesthat can be bound by RNA recognition motif (RRM) domains (e.g., U1A),sequences that can be bound by double stranded RNA binding domains(dsRBD) (e.g., Staufen), sequences that can be bound PAZ domains (e.g.,PAZ, Argonaute), sequences that can be bound by PIWI domains (e.g.,PIWI, MILI, MIWI, Argonaute), and the like. Some examples of nucleicacid-binding protein binding sites can include, for example, sequencesthat can be bound by DNA-binding proteins such as zinc fingers, ahelix-turn-helix domain, a zinc finger domain, a leucine zipper (bZIP)domain, a winged helix domain, a winged helix turn helix domain, ahelix-loop-helix domain, a HMG-box domain, a Wor3 domain, animmunoglobulin domain, a B3 domain, a TALE domain, and the like.

The nucleic acid can comprise one or more nucleic acid-binding proteinbinding sites. The nucleic acid can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more nucleic acid-binding protein binding sites. The one or morenucleic acid-binding protein binding sites may be the same. The one ormore nucleic acid-binding protein binding sites may be different. Forexample, the nucleic acid can comprise a Csy4 binding site and a MS2binding site. The one or more nucleic acid-binding protein binding sitescan be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250,300, 350, 400, 450, 500 or more nucleotides. In some embodiments, the3′-most nucleic acid-binding protein binding site can be bound by atandem fusion polypeptide of the disclosure.

Tandem Fusion Polypeptide

In some embodiments, the method of the disclosure provides for binding aplurality of nucleic acids to a tandem fusion polypeptide. A tandemfusion polypeptide can comprise a plurality of nucleic acid bindingproteins fused together in one polypeptide chain. A tandem fusionpolypeptide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleicacid-binding proteins. Nucleic acid-binding proteins of the tandemfusion polypeptide can bind to the nucleic acid-binding protein bindingsites of the nucleic acids of the disclosure. Examples of nucleicacid-binding proteins can include MS2, U1A, boxB sequence bindingproteins (e.g., zinc fingers), eIF4A, Staufen, PAZ, Argonaute, PIWI,MILI, MIWI, zinc fingers, a helix-turn-helix domain, a zinc fingerdomain, a leucine zipper (bZIP) domain, a winged helix domain, a wingedhelix turn helix domain, a helix-loop-helix domain, a HMG-box domain, aWor3 domain, an immunoglobulin domain, a B3 domain, a TALE domain, andthe like. In some embodiments, the nucleic acid-binding protein is anRNA-binding protein. The RNA-binding protein can be a member of a CRISPRsystem. In some embodiments, the RNA-binding protein can be a member ofthe Cas5 or Cas6 family of proteins. In some embodiments, theRNA-binding protein can be Csy4, Cas5, Cas6, or any combination thereof.In some embodiments, the nucleic acid-binding protein is a DNA-bindingprotein (e.g., a zinc finger).

In some instances, the nucleic acid-binding proteins are separated by alinker. A linker can comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100 or more amino acids.

A tandem fusion polypeptide can comprise a non-native sequence (e.g.,peptide affinity tag). The non-native sequence can comprise a nuclearlocalization signal (NLS) that can direct the tandem fusion polypeptideto a subcellular location (e.g., nucleus).

Each nucleic acid-binding protein of the tandem fusion polypeptide cancomprise its own non-native sequence. The non-native sequence of eachnucleic acid-binding protein can be the same. The non-native sequence ofeach nucleic acid-binding protein can be different. The non-nativesequence of some of the nucleic acid-binding proteins of the tandemfusion polypeptide can be the same and the non-native sequence of someof the nucleic acid-binding proteins of the tandem fusion polypeptidecan be different.

In some instances, the methods of the disclosure can provide for forminga complex comprising a tandem fusion polypeptide and a plurality ofnucleic acids of the disclosure. Formation of the complex can comprisethe nucleic acid-binding proteins of the tandem fusion polypeptidebinding to their cognate nucleic acid-binding protein binding sequencein the nucleic acids of the disclosure. For example, astoichiometrically deliverable nucleic acid comprising a Csy4 bindingsite, can bind to the Csy4 protein subunit in the tandem fusion protein.The complex can be formed outside of cells (e.g., in vitro). The complexcan be formed in cells (e.g., in vivo). When a complex is formed invitro it can be introduced into a cell by, for example, transfection,transformation, viral transduction, electroporation, injection, and thelike.

The methods of the disclosure provide for therapeutic delivery ofmultiple nucleic acids both in vivo, in vitro, and ex vivo. Thedelivered nucleic acids can be used to treat a disease. For example, thedelivered nucleic acids can be used in gene therapy and/or can integrateinto the genome of the cell, thereby providing a therapeutic outcome. Atherapeutic outcome can refer to increase or decrease in the levels of aprotein, nucleic acid, or any biological molecule related to a diseasesuch as a degradation product, small molecule, and/or ion. For example,a therapeutic outcome can comprise increasing the levels of ananti-inflammatory gene, or decreasing the levels of a protein in apathway related to a disease. A therapeutic outcome can refer to aphysiological effect. Physiological effects can include, morphologicalchanges, metabolic changes, and/or structural changes in a cell. Atherapeutic outcome can refer to changes in the modifications of aprotein and/or nucleic acid, such as glycosylation, acetylation,methylation, demethylation, depurination, ubiquitinylation, and thelike.

A therapeutic outcome can be measured by changes in the genetic makeupof the cell, the levels of biomolecules of interest in the cell, and/orthe physiological changes in the cell. Measurements can be made usingmolecular biology techniques such as spectroscopy, spectrometry,sequencing, ELISA, microscopy, and/or x-ray crystallograhpy.Measurements can be made using animal models, such as mouse, rats, dogs,and primates. For example, genetically modified cells of the disclosurecan be introduced into mice and assessed for biological andphysiological changes such as, for example, the ability to metastasizeand/or differentiate.

Spacers for Blood Disorders

This disclosure provides for compositions, methods and kits, for geneticengineering of hematopoietic stem cells (HSCs).

Compositions

A HSC can comprise a site-directed polypeptide (e.g., Cas9).

A HSC can comprise a nucleic acid-targeting nucleic acid. The nucleicacid-targeting nucleic acid of the disclosure can target a gene involvedin a genetic disorder. Table 1 lists exemplary genes that are involvedin disorders. The genes listed in Table 1 can be genes that can betargeted by a nucleic acid-targeting nucleic acid. The nucleicacid-targeting nucleic acid of the disclosure can comprise a spacer thatcan target a gene listed in Table 1.

Table 2 depicts exemplary spacers of the nucleic acid-targeting nucleicacids of the disclosure. Each spacer of Table 2 can be a spacer that canbe inserted into a nucleic acid-targeting nucleic acid. Exemplaryspacers are companied by the name of the disorder and the gene involvedin the disorder that is targeted by the spacers.

TABLE 2 Spacers of blood disorders. Gene Spacer Disease Arylsulfatase AAGGGTTTATTTTCTTACGCT Metachromatic Leukodystrophy Wiskott-AldrichCCGAGGTCCCTAGTCCGGAA Wiskott-Aldrich Syndrome Syndrome proteinATP-binding CGGAGGTGGGCGGAGCCTCC Adrenoleukodystrophy cassette D1C-C chemokine AGCTTTCTCGTCTGGGTATT Human Immunodeficiency Virusreceptor type 5 Hemoglobin beta GAAAATAAATGTTTTTTATT Beta-thalassemiasubunit Interleukin-2 ACAGAAACTTTATTTCTCAT X-linked Severe Combined IDrecetor subunit gamma Cystinosin CTTTGGGAGGCCGAGGCGGG MLSD cystinosisRibosomal protein TTTTAGAAACAGTATGAGAT Diamon-Blackfan anemia S19Fanconi anemia ATGCACAAAATAAACAGCAG Fanconi Anemia complimentationgroup B Shwachman- GAGTTAGTTCACATCTACAG Shwachman-Bodian-DiamondBodian-Diamond syndrome syndrome gene

Methods

The disclosure provides for methods for introducing a nucleicacid-targeting nucleic acid and site-directed polypeptide into an HSC.In some embodiments, the HSC is extracted from a patient prior tointroduction. The extracted HSC can be purified (e.g., by apheresis).The site-directed polypeptide and/or the nucleic acid-targeting nucleicacid can be introduced into an HSC that has been purified. Thesite-directed polypeptide and/or the nucleic acid-targeting nucleic acidcan be introduced into an HSC that has not been purified. Theintroduction can occur in an HSC in vitro (e.g., outside of the patient,extracted cell). In some instances, the introduction occurs in an HSC invivo (e.g., inside of a patient, unextracted cell).

Introduction of the site-directed polypeptide and/or the nucleicacid-targeting nucleic acid of the disclosure can occur by for example,viral transduction, transfection, electroporation, optical transfectionand/or chemical transfection.

Once introduced into a HSC, the nucleic acid-targeting nucleic acid ofthe disclosure and the site-directed polypeptide can form a complex. Thecomplex can be guided to a target nucleic acid (e.g., the genes listedin Table 3) by the nucleic acid-targeting nucleic acid. The nucleicacid-targeting nucleic acid can hybridize with the target nucleic acid.The site-directed polypeptide can modify the target nucleic acid (e.g.,by cleaving the target nucleic acid).

In some instances, the modified target nucleic acid comprises adeletion. In some instances, the modified target nucleic acid comprisesan insertion of a donor polynucleotide. A donor polynucleotide, aportion of a donor polynucleotide, a copy of a donor polynucleotide or aportion of a copy of a donor polynucleotide can be inserted into atarget nucleic acid. The method can be performed using any of thesite-directed polypeptides, nucleic acid-targeting nucleic acids, andcomplexes of site-directed polypeptides and nucleic acid-targetingnucleic acids as described herein.

TABLE 3 List of Genes Involved in Diseases Name of Disease Gene(s)Metachromatic leukodystrophy (MLD) Arylsulfatase A Wiskott-Aldrichsyndrome (WAS) Wiskott-Aldrich Syndrome protein Wiskott-Aldrich syndrome(WAS) Leukosialin Neutropenia Wiskott-Aldrich Syndrome proteinAdrenoleukodystrophy ATP-binding cassette D1 Human ImmunodeficiencyVirus (HIV) C-C chemokine receptor type 5 Beta-thalassemia hemoglobinsubunit beta Sickle-cell anemia hemoglobin subunit beta X-linked SevereCombined Immunodeficiency (X- Interleukin-2 receptor subunit gamma SCID)Multisystemic Lysosomal Storage Disorder Cystinosin cystinosisDiamond-Blackfan anemia Ribosomal protein S19 Fanconi Anemia Fanconianemia complementation groups A, B and C Shwachman-Bodian-Diamondsyndrome SBDS gene Gaucher's disease Glucocerebrosidase Hemophilia AAnti-hemophiliac factor OR Factor VIII Hemophilia B Christmas factor,Serine protease, Factor IX Adenosine deaminase deficiency (ADA-SCID)Adenosine deaminase GM1 gangliosidoses beta-galactosidease Glycogenstorage disease type II, Pompe disease, acid alpha-glucosidase acidmaltase deficiency Niemann-Pick disease, SMPD1-associated (TypesSphingomyelin phosphodiesterase 1 OR A and B) acid sphingomyelinaseKrabbe disease, globoid cell leukodystrophy, Galactosylceramidase ORgalactosylceramide lipidosis galactercerebrosidease Multiple Sclerosis(MS) Human leukocyte angitens DR-15, DQ-6, DRB1

Computational Methods

The disclosure provides for computational methods to identify spacersfor nucleic acid-targeting nucleic acids. The computational method cancomprise scanning the nucleic acid sequence of a genome for aprotospacer adjecent motif. Upon finding a protospacer adjacent motif,the program can automatically count between 10-30 nucleotides upstreamof the protospacer adjacent motif. The 10-30 nucleotides upstream of theprotospacer adjacent motif can constitute a putative spacer sequence. Inother words, the 10-30 nucleotides upstream of the protospacer adjcentmotif in the genome can correspond to a target nucleic acid, and asequence complementary to the target nucleic acid can be referred to asa spacer.

The program can test every sequence iteration of the putative spacersequence to ascertain how effective the sequence will be as a spacer ina nucleic acid-targeting nucleic acid. For example, the program can takeeach iteration of the putative spacer sequence and perform an in silicosecondary structure prediction on the sequence. The secondary structureprediction can comprise appending the putative spacer sequences to anucleic acid-targeting nucleic acid backbone (e.g., the nucleicacid-targeting nucleic acid without the spacer). The secondary structureprediction can perform a secondary structure prediction analysis of thedocked putative spacer sequence in the nucleic acid-targeting nucleicacid backbone. Secondary structure prediction analysis can comprise, forexample, predicting which nucleotides may form duplexes, hairpins, whichnucleotides are unstructured, and/or which nucleotides may be unpaired.

The computational method can comprise implementing a folding test oneach putative spacer sequence that has undergone secondary structureprediction analysis. The folding test can comprise in silico folding ofthe nucleic acid-targeting nucleic acid comprising the putative spacersequence. The nucleic acid-targeting nucleic acid and putative spacersequence can either pass or fail the folding test.

To pass the folding test, the secondary structure of the backbonenucleic acid-targeting nucleic acid may need to be conserved, less than5, 4, 3, 2, or 1 nucleotide in the spacer hybridize with nucleotidesoutside of the spacer, and other second structure in the spacer iscontained within the spacer.

Seamless Reporter Selection

General Overview

This disclosure describes methods, compositions, systems and kits forgenetic modification of cells and selection of such genetically modifiedcells by seamless incorporation, detection and excision of a reporterelement. In some embodiments of the disclosure, a donor polynucleotidecan comprise a nucleic acid to be introduced to a cell genome (herecalled the genetic element of interest) as well as a nucleic acidsequence encoding a reporter element (e.g. GFP), a site-directedpolypeptide and two nucleic acid-targeting nucleic acids. Either thesite-directed polypeptide, the nucleic acid-targeting nucleic acids,and/or all three may be controlled by an inducible promoter. Asite-directed polypeptide and a nucleic acid-targeting nucleic acid mayform a complex which can target a site in the cell genome byhybridization of the nucleic acid targeting nucleic acid to a targetnucleic acid in the genome. The site-directed polypeptide of the complexmay cleave the target nucleic acid. The donor polynucleotide can beinserted into the cleaved target nucleic acid. After introduction of adouble strand break (or single strand break) at the target site in thepresence of the donor polynucleotide, the population of recipient cellsmay be screened for the presence of the reporter molecule as a proxy forthe presence of the genetic element of interest. After isolation ofreporter molecule-containing cells, the reporter element can be excisedby induction of the site-directed polypeptide and/or nucleicacid-targeting nucleic acid expression. The nucleic acid-targetingnucleic acids can target the 5′ and 3′ ends of the reporter element andcan result in the excision of the reporter element. The method can beperformed using any of the site-directed polypeptides, nucleicacid-targeting nucleic acids, and complexes of site-directedpolypeptides and nucleic acid-targeting nucleic acids as describedherein.

FIG. 18 depicts an exemplary embodiment of the methods of thedisclosure. A nucleic acid can comprise a plurality of genetic elements1805/1810. The genetic elements 1805 and 1810 can be, for example,genes, non-coding nucleic acids, introns, exons, DNA and/or RNA. Thegenetic elements 1805 and 1810 can be parts of the same gene. In betweenthe genetic elements can be a target nucleic acid 106 suitable forgenetic engineering. A site-directed polypeptide and a nucleicacid-targeting nucleic acid of the disclosure can form a complex whichcan target 1815 the target nucleic acid 1806. The site-directedpolypeptide of the complex can cleave 1820 the target nucleic acid 1806.A donor polynucleotide can be inserted 1825 into the cleaved targetnucleic acid 1806. The donor polynucleotide can comprise a geneticelement of interest 1830. The genetic element of interest 1830 can be agene. The donor polynucleotide can also comprise a reporter element1835. The donor polynucleotide can also comprise a polynucleotidesequence encoding a site-directed polypeptide and one or more nucleicacid-acid targeting nucleic acid. In some instances, the polynucleotideencoding the site-directed polypeptide and the one or more nucleicacid-targeting nucleic acids encodes two nucleic acid-targeting nucleicacids. The polynucleotide sequence encoding the site-directedpolypeptide and the nucleic acid targeting nucleic acid can be operablylinked to an inducible promoter. Insertion of the donor polynucleotideinto the target nucleic acid 1806 can result in the expression of thereporter element 1835. The reporter element 1835 can be used as a way toselect cells that comprise the donor polynucleotide.

FIG. 19 depicts an exemplary embodiment for the removal of the reporterelement 1915 from the target nucleic acid. A target nucleic acid cancomprise a plurality of genetic elements 1905/1920. The reporter element1915 can be fused to a genetic element of interest 1910. Expression ofthe reporter gene 1915 can be induced which can result in the productionof a site-directed polypeptide and one or more nucleic acid-targetingnucleic acids. The site-directed polypeptide can form complexes with thenucleic acid-targeting nucleic acids. The complexes can be guided to thereporter element 1915 by the nucleic acid-targeting nucleic acid of thecomplex. One of the two nucleic acid-targeting nucleic acids can target1925 the 5′ end of the reporter element 1915. One of the two nucleicacid-targeting nucleic acids can target 1930 the 3′ end of the reporterelement 1915. The targeted ends of the reporter element 1915 can becleaved by the site-directed polypeptide of the complex, therebyexcising 1935 the reporter element 1915. The target nucleic acid cancomprise the genetic element of interest 1910 portion of the donorpolynucleotide. The nucleic acid-targeting nucleic acids can be designedsuch that the donor polynucleotide is excised (including the geneticelement of interest).

Methods

The present disclosure provides for methods of selecting cells using areporter element and excision of the reporter element. A site-directedpolypeptide, an endoribonuclease, a nucleic acid targeting nucleic acid,a donor polynucleotide and/or a nucleic acid-targeting nucleic acid canbe introduced into a cell. The donor polynucleotide may include one ormore genetic elements of interest. The donor polynucleotide may includeone or more reporter elements. The donor polynucleotide includes one ormore genetic elements of interest and one or more reporter elements.More than one site-directed polypeptide, endoribonuclease, donorpolynucleotide and and/or nucleic acid-targeting nucleic acid can beintroduced into a cell. In some instances, the cell already expresses asite-directed polypeptide, and/or a nucleic acid-targeting nucleic acid.In some instances, the site-directed polypeptide, and/or nucleic acidtargeting nucleic acid are encoded on a plasmid. In some instances, thesite-directed polypeptide, and/or nucleic acid targeting nucleic acid isencoded on more than one plasmid. In some instances, more than onesite-directed polypeptide or nucleic acid encoding a site-directedpolypeptide is introduced into the cell. In some instances, the cell isa cell lysate.

Introduction can occur by any means to introduce a nucleic acid into acell such as viral or bacteriophage infection, transfection,conjugation, protoplast fusion, lipofection, electroporation, calciumphosphate transfection, polyethyleneimine (PEI)-mediated transfection,DEAE-dextran mediated transfection, liposome-mediated transfection,particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.The vector can be transiently expressed in the host cell. The vector canbe stably expressed in the host cell (e.g., by stably integrating intothe genome of the host cell).

A nucleic acid targeting nucleic acid can bind to a nucleic acidcharacterized by a particular target sequence and/or any sequencehomologous to a particular sequence. The target sequence can be part orall of a gene, a 5′ end of a gene, a 3′ end of a gene, a regulatoryelement (e.g. promoter, enhancer), a pseudogene, non-coding DNA, amicrosatellite, an intron, an exon, chromosomal DNA, mitrochondrial DNA,sense DNA, antisense DNA, nucleoid DNA, chloroplast DNA or RNA amongother nucleic acid entities.

The site-directed polypeptide can cleave the target nucleic acid boundby a nucleic acid targeting nucleic acid. The site-directed polypeptidemay not cleave the target nucleic acid. In some instances, anendoribonuclease cleaves the target nucleic acid. The endoribonucleasecan be encoded by the vector. The endoribonuclease can be encoded by thedonor polynucleotide. The endoribonuclease can be present in the cell.Expression of the endoribonuclease and/or site-directed polypeptide canbe induced by a conditional promoter. A donor polynucleotide can beincorporated in the target nucleic acid at the site where it wascleaved.

Excision

The methods disclosed herein may further comprise excision of all, someor none of the reporter element. A first nucleic acid-targeting nucleicacids of the reporter element can target the 5′ end of the reporterelement. A second nucleic acid-targeting nucleic acids of the reporterelement can target the 3′ end of the reporter element. A nucleicacid-targeting nucleic acid can target both the 5′ and 3′ ends of thereporter element. A nucleic acid-targeting nucleic acid can target twosequences in the reporter element and/or donor polynucleotide. The twotarget sequences can be at least about 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98, 99 or 100% identical. The two target sequences canbe at most about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identical. When the nucleic acid-targeting nucleic acids of thereporter element are expressed, they may form a complex with asite-directed polypeptide and target the 5′ and 3′ ends of the reporterelement by hybridizing to a complementary region on the 5′ and 3′ endsof the reporter element. Hybridization of the complex with the reporterelement can result in cleavage of all, some or none of the reporterelement. The cleaved nucleic acid can be rejoined by, for example,non-homologous end joining. The rejoined nucleic acid may not introducea deletion or insertion. The rejoined nucleic acid may introduce adeletion or insertion. The cleaved nucleic acid can be rejoined by, forexample, homologous recombination. Homologous recombination can be usedto rejoin a cleaved nucleic acid when the target nucleic acid sites aresubstantially identical.

Screening

The methods disclosed herein may further comprise excising a reporterelement from a selected cell, thereby forming a second cell; andscreening the second cell. Screening may comprise screening for theabsence of all or some of the reporter element. Screening can includefluorescene activate cell-sorting (FACS), wherein cells expressing afluorescent protein encoded for by the reporter element are separatedfrom cells that do not express a fluorescent protein. Cells may becontacted with fluorescent protein, fluorescent probe or fluorochromeconjugated antibodies that bind proteins encoded for by the reporterelement or genetic element and subsequently selected by FACS.Fluorochromes can include but are not limited to Cascade Blue, PacificBlue, Pacific Orange, Lucifer yellow, NBD, R-Phycoerythrin (PE), PE-Cy5conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX,Fluorescein, BODIPY-FL, TRITC, Texas Red, Allophycocyanin, APC-Cy7conjugates (PharRed), various Alexa Fluor dyes, Cy2, Cy3, Cy3B, Cy3.5,Cy5, Cy5.5, Cy7, various DyLights, Y66H, Y66F, EBFP, EBFP2, Azurite,GFPuv, T-Sapphire, TagBFP, Cerulean, mCFP, ECFP, CyPet, Y66W,dKeima-Red, mKeima-Red, TagCFP, AmCyanl, mTFP1, S65A, Midoriishi-Cyan,GFP, Turbo GFP, TagGFP, TagGFP2, AcGFP1, S65L, Emerald, S65T, S65C,EGFP, Azami-Green, ZaGreenl, Dronpa-Green, TagYFP, EYFP, Topaz, Venus,mCitirine, YPet, Turbo YFP, PhiYFP, PhiYFPm, ZaYellowl, mBanana,Kusabira-Orange, mOrange, mOrane2, mKO, TurboRFP, tdTomato,DsRed-Express2, TagRFP, DsRed monomer, DsRed2, mStrawberry, Turbo FP602,AsRed2, mRFP1, J-Red, mCherry, HcRedl, mKate2, Katushka, mKate,TurboFP635, mPlum, mRaspberry, mNeptune, E2-Crimson.

Cells may be contacted with antibodies that bind peptide affinity tagsencoded for by the reporter element or genetic element and subsequentlycan be selected by immunomagnetic beads which recognize the antibodies.Screening may comprise staining cells by adding X-gal when the reporterelement or genetic element encodes b-galactosidase. Screening maycomprise manual sorting (e.g. diluting cell suspensions) and microscopy(e.g. fluorescence microscopy). Screening may comprise high-contentscreening.

Reporter elements may encode drug resistance genes, thereby allowing forselection of cells containing the reporter element by the addition ofdrugs, the drugs killing the cells that do not express the reporterelement. Such drug can include, but are not limited to erythromycin,clindamycin, chloramphenicol, gentamicin, kanamycin, streptomycin,tetracycline, the combination quinupristin-dalfopristin, enrofloxacin,vancomycin, oxacillin, penicillin, sulfonamide sulfisoxazole,trimethoprim, methoinine sulphoximine, methotrexate, puromycine,blasticidin, histidinol, hygromycin, zeocin, bleomycin and neomycin.

Libraries

The present disclosure provides for a library of expression vectorscomprising donor polynucleotides. In some embodiments, the library cancomprise expression vectors comprising polynucleotide sequences encodingfor differing genetic elements of interest but the same reporterelements. In some embodiments, the library can comprise expressionvectors comprising polynucleotide sequences encoding for differinggenetic elements of interest and differing reporter elements. Reporterelements may differ in their nucleic acid targeting sequences (crRNA andtracrRNA). Reporter elements may differ in their reporter genes (e.g.genes encoding fluorescent proteins). The present disclosure providesfor methods of using the library to generate a plurality of geneticallymodified cells. The present disclosure provides for methods of using thelibrary for a high throughput genetic screen. These libraries can allowfor analyzing large numbers of individual genes to infer gene function.Libraries can comprise from about 10 individual members to about 10¹²individual members; e.g. a library can comprise from about 10 individualmembers to about 10² individual members, from about 10² individualmembers to about 10³ individual members, from about 10³ individualmembers to about 10⁵ individual members, from about 10⁵ individualmembers to about 10⁷ individual members, from about 10⁷ individualmembers to about 10⁹ individual members, or from about 10⁹ individualmembers to about 10¹² individual members.

Modifying Cells (Transfection/Infection)

Methods of disclosure provide for selection of cells comprising thedonor polynucleotide. In some embodiments, a method can involvecontacting a target nucleic acid or introducing into a cell (or apopulation of cells) one or more nucleic acids comprising nucleotidesequences encoding a nucleic acid-targeting nucleic acid, asite-directed polypeptide, and/or donor polynucleotide of thedisclosure. Methods for introducing a nucleic acid into a cell caninclude viral or bacteriophage infection, transfection, conjugation,protoplast fusion, lipofection, electroporation, calcium phosphateprecipitation, polyethyleneimine (PEI)-mediated transfection,DEAE-dextran mediated transfection, liposome-mediated transfection,particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like. Insome embodiments, contacting a target nucleic acid or introducing into acell (or a population of cells) one or more nucleic acids may notcomprise viral infection. In some embodiments, contacting a targetnucleic acid or introducing into a cell (or a population of cells) oneor more nucleic acids may not comprise bacteriophage infection. In someembodiments, contacting a target nucleic acid or introducing into a cell(or a population of cells) one or more nucleic acids may not comprisetransfection.

Engineered Nucleic Acid-Targeting Nucleic Acids

Engineered P-Domains

A nucleic acid-targeting nucleic acid can be engineered (e.g., comprisemodifications). An engineered nucleic acid-targeting nucleic acid canrefer to any of the engineered nucleic acid-targeting nucleic acids asdescribed herein. For example, an engineered nucleic acid-targetingnucleic acid can comprise a minimum CRISPR repeat, a minimum tracrRNA,and a 3′ tracrRNA. A P-domain of a nucleic acid-targeting nucleic acidcan interact with region of a site-directed polypeptide. A P-domain caninteract with a plurality of regions of a site-directed polypeptide. AP-domain can interact with a plurality of regions of a site-directedpolypeptide wherein at least one of the regions interacts with a PAM ina protospacer adjacent motif. Examples of these regions can includeamino acids 1096-1225, and 1105-1138 of Cas9 in S. pyogenes.

A modification can be introduced into the P-domain. A P-domain cancomprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30or more adjacent nucleotides. A P-domain can comprise at most about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 or more adjacentnucleotides. A P-domain can start one nucleotide 3′ of the last pairednucleotide in the duplex comprising the minimum CRISPR repeat and theminimum tracrRNA sequence. A P-domain can start at least about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 or more nucleotides 3′ of thelast paired nucleotide in the duplex comprising the minimum CRISPRrepeat and the minimum tracrRNA sequence. A P-domain can start at mostabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 or morenucleotides 3′ of the last paired nucleotide in the duplex comprisingthe minimum CRISPR repeat and the minimum tracrRNA sequence.

An engineered P-domain can comprise at least about 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or more mutations. An engineered P-domain can comprise atmost about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations. Themutations can be adjacent to one another (e.g., sequential). Themutations can be separated from one another. The mutations can beseparated by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or morenucleotides. The mutations can be separated by at least about 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 or more nucleotides. Mutations to a nucleicacid-targeting nucleic acid can comprise insertions, deletions, andsubstitutions of nucleotides in the nucleic acid-targeting nucleic acid.

In some instances, an engineered nucleic acid-targeting nucleic acidcomprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30% or morenucleotide identity and/or similarity to a wildtype nucleicacid-targeting nucleic acid. In some instances, an engineered nucleicacid-targeting nucleic acid comprises at least about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30% or more nucleotide identity and/or similarity to awildtype nucleic acid-targeting nucleic acid.

In some instances, a CRISPR nucleic acid portion of the engineerednucleic acid-targeting nucleic acid comprises at most about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30% or more nucleotide identity and/orsimilarity to a wildtype CRISPR nucleic acid. In some instances, aCRISPR nucleic acid portion of the engineered nucleic acid-targetingnucleic acid comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30% or more nucleotide identity and/or similarity to a wildtype CRISPRnucleic acid.

A tracrRNA nucleic acid portion of the engineered nucleic acid-targetingnucleic acid can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30% or more nucleotide identity and/or similarity to a wildtypetracrRNA nucleic acid. A tracrRNA nucleic acid portion of the engineerednucleic acid-targeting nucleic acid can comprise at least about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30% or more nucleotide identity and/orsimilarity to a wildtype tracrRNA nucleic acid.

The modifications in the P-domain can be such that the engineerednucleic acid-targeting nucleic acid is newly configured to hybridize toa new PAM sequence in a target nucleic acid. The modification to theP-domain can be complementary to the PAM in the target nucleic acid. Themodification to the P-domain can comprise the reverse complement of thePAM in the target nucleic acid. The new PAM can comprise at least about1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides. The new PAM cancomprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or morenucleotides. Modifications in the P domain can occur in concert withmodifications to a P-domain binding and PAM-binding region of asite-directed polypeptide. These modifications can be compensatory,wherein a modified P-domain is specifically modified to bind to amodified site-directed polypeptide, wherein the modification enables thesite-directed polypeptide to bind to the engineered P-domain withgreater specificity.

An engineered P-domain can be engineered to bind to a new PAM (e.g., theengineered P-domain can hybridize to a new PAM). The new PAM (e.g.,different PAM), can be bimodal (i.e., a bimodal PAM can comprise twoseparate regions of the PAM). The two separate regions of a bimodal PAMcan be separated by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore nucleotides. The two separate regions of a bimodal PAM can beseparated by at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or morenucleotides.

An engineered P-domain can be be engineered to bind to a new PAM (e.g.,different PAM), wherein the new PAM is trimodal. A trimodal PAM cancomprise three separate regions of a PAM sequence (e.g., three separateregions that can be used in targeting a nucleic acid-targeting nucleicacid to a target nucleic acid). The three separate regions of a trimodalPAM can be separated by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10or more nucleotides. The three separate regions of a trimodal PAM can beseparated by at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or morenucleotides.

An engineered nucleic acid-targeting nucleic acid can comprise at leasttwo hairpins. A first hairpin can comprise a duplex between the minimumCRISPR repeat and the minimum tracrRNA sequence. The second hairpin canbe downstream of the first hairpin. The second hairpin can start atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides downstream ofthe last paired nucleotide of the first duplex. The second hairpin canstart at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotidesdownstream of the last paired nucleotide of the first duplex. The secondhairpin can comprise an engineered P-domain.

The engineered P-domain of the second hairpin can be located on one sideof the duplex hairpin. The engineered P-domain of the second hairpin canbe located on both sides of the duplex hairpin. The engineered P-domainof the second hairpin can comprise at least about 1, 2, 3, 4, 5, 10, or20% of the nucleotides in the second hairpin. The engineered P-domain ofthe second hairpin can comprise at most about 1, 2, 3, 4, 5, 10, or 20%of the nucleotides in the second hairpin.

The second hairpin can comprise a tracrRNA (e.g., the mid-tracrRNA, or3′ tracrRNA of a nucleic acid-targeting nucleic acid). The secondhairpin can comprise at least about 1, 5, 10, 15, 20, 25, 30, 35, 40,45, or 50% or more identity to a tracrRNA. The second hairpin cancomprise at most about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% ormore identity to a tracrRNA.

The second hairpin comprising the engineered P-domain can be configuredto de-duplex (e.g., melt, unwind). The second hairpin can de-duplex whenin contact with a target nucleic acid. The second hairpin can de-duplexwhen in contact with a protospacer adjacent motif of a target nucleicacid.

In some instances, an engineered P-domain can be configured to hybridizeto a region in a nucleic acid-targeting nucleic acid (e.g., the samenucleic acid-targeting nucleic acid comprising the engineered P-domain),and the engineered P-domain can be configured to hybridize to a targetnucleic acid. In other words, the engineered P-domain can comprise aswitchable sequence, in which in some instances, the P-domainishybridized to the nucleic acid-targeting nucleic acid, thereby forming ahairpin, and in some instances the P-domain is hybridized to a PAM in atarget nucleic acid.

An engineered nucleic acid-targeting nucleic acid comprising a modifiedP-domain can be engineered to bind to a PAM with a lower dissociationconstant than a nucleic acid-targeting nucleic acid that does notcomprise a modified P-domain (e.g., wild-type nucleic acid-targetingnucleic acid). An engineered nucleic acid-targeting nucleic acidcomprising a modified P-domain can be engineered to bind to a PAM with adissociation constant at least about 10, 50, 100, 150, 200, 250, 300,350, 400, 450, 500, 550 or 600% or more lower or higher than a nucleicacid-targeting nucleic acid that does not comprise a modified P-domain.

An engineered nucleic acid-targeting nucleic acid comprising a modifiedP-domain can be engineered to bind to a PAM with a dissociation constantat most about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550or 600% or more lower or higher than a nucleic acid-targeting nucleicacid that does not comprise a modified P-domain. An engineered nucleicacid-targeting nucleic acid comprising a modified P-domain can beengineered to bind to a PAM with a dissociation constant at least about1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold,40-fold, or 50-fold or more lower or higher than a nucleicacid-targeting nucleic acid that does not comprise a modified P-domain.An engineered nucleic acid-targeting nucleic acid comprising a modifiedP-domain can be engineered to bind to a PAM with a dissociation constantat most about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold,30-fold, 40-fold, or 50-fold or more lower or higher than a nucleicacid-targeting nucleic acid that does not comprise a modified P-domain.

An engineered nucleic acid-targeting nucleic acid comprising a modifiedP-domain can be engineered to bind to a PAM with greater specificitythan a nucleic acid-targeting nucleic acid that does not comprise amodified P-domain (e.g., wild-type nucleic acid-targeting nucleic acid).Greater specificity can refer to a reduction in off-target binding(e.g., binding of the nucleic acid-targeting nucleic acid to anincorrect PAM or PAM-like sequence). For example, an engineered nucleicacid-targeting nucleic acid comprising a modified P-domain can beengineered to reduce non-specific binding by at least about 10, 50, 100,150, 200, 250, 300, 350, 400, 450, 500, 550 or 600% or more than anucleic acid-targeting nucleic acid that does not comprise a modifiedP-domain. An engineered nucleic acid-targeting nucleic acid comprising amodified P-domain can be engineered to reduce non-specific binding by atmost about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or600% or more than a nucleic acid-targeting nucleic acid that does notcomprise a modified P-domain. An engineered nucleic acid-targetingnucleic acid comprising a modified P-domain can be engineered to reducenon-specific binding by at least about 1-fold, 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold or than a nucleicacid-targeting nucleic acid that does not comprise a modified P-domain.An engineered nucleic acid-targeting nucleic acid comprising a modifiedP-domain can be engineered to reduce non-specific binding by at mostabout 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold,40-fold, or 50-fold or more than a nucleic acid-targeting nucleic acidthat does not comprise a modified P-domain.

Engineered Bulges

An engineered nucleic acid-targeting nucleic acid can be engineered suchthat a modification can be introduced into the bulge region of thenucleic acid-targeting nucleic acid. A bulge is a typical nucleic acidfeature that comprises unpaired nucleotides. A bulge can compriseunpaired nucleotides on each strand of the duplex that comprises thebulge. In other words, a bulge can comprise an unpaired nucleotide onthe minimum CRISPR repeat strand of the duplex and an unpairednucleotide on the minimum tracrRNA sequence strand of the duplex.

A bulge can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ormore unpaired nucleotides on a first strand of a duplex in a nucleicacid-targeting nucleic acid (i.e., the minimum CRISPR repeat strand ofthe duplex comprising the minimum CRISPR repeat and minimum tracrRNAsequence). A bulge can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 or more unpaired nucleotides on a first strand of a duplex in anucleic acid-targeting nucleic acid (i.e., the minimum CRISPR repeatstrand of the duplex comprising the minimum CRISPR repeat and minimumtracrRNA sequence). A bulge can comprise at least about 1, 2, 3, 4, 5,6, 7, 8, 9 or 10 or more unpaired nucleotides on a second strand of aduplex in a nucleic acid-targeting nucleic acid (i.e., the minimumtracrRNA sequence strand of the duplex comprising the minimum CRISPRrepeat and minimum tracrRNA sequence). A bulge can comprise at mostabout 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more unpaired nucleotides on asecond strand of a duplex in a nucleic acid-targeting nucleic acid(i.e., the minimum tracrRNA sequence strand of the duplex comprising theminimum CRISPR repeat and minimum tracrRNA sequence). A bulge cancomprise one unpaired nucleotide on the minimum CRISPR RNA sequence and3 unpaired nucleotides on the minimum tracrRNA sequence strand.

The nucleotides adjacent to an unpaired nucleotide can be a nucleotidethat forms a wobble base pairing interaction. Wobble base pairinginteractions can include guanine-uracil, hypoanthine-uracil,hypoxanthine-adenine, and hypoxanthine-cytosine. At least 1, 2, 3, 4, or5 or more nucleotides adjacent to an unpaired nucleotide can form awobble pairing. At most 1, 2, 3, 4, or 5 or more nucleotides adjacent toan unpaired nucleotide can form a wobble pairing.

An engineered bulge can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 or more mutations. An engineered bulge can comprise at mostabout 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations. The mutationscan be adjacent to one another (e.g., sequential). The mutations can beseparated from one another. The mutations can be separated by at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. Themutations can be separated by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 or more nucleotides. Mutations to a nucleic acid-targeting nucleicacid can comprise insertions, deletions, and substitutions ofnucleotides in the nucleic acid-targeting nucleic acid.

A bulge of an engineered nucleic acid-targeting nucleic acid cancomprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30% or morenucleotide identity and/or similarity to a wildtype nucleicacid-targeting nucleic acid. A bulge of an engineered nucleicacid-targeting nucleic acid can comprise at least about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30% or more nucleotide identity and/or similarity toa wildtype nucleic acid-targeting nucleic acid.

One strand of the bulge can be mutated and the other strand is notmutated. In other words, in some instances, the sequence of the bulge onthe minimum CRISPR RNA strand is the same as a wild-type nucleicacid-targeting nucleic acid, and the sequence of the bulge on theminimum tracrRNA sequence is mutated. In other words, the sequence ofthe bulge on the minimum CRISPR RNA strand is mutated, and the sequenceof the bulge on the minimum tracrRNA sequence is the same as a wild-typenucleic acid-targeting nucleic acid.

The modifications in the bulge can be such that the engineered nucleicacid-targeting nucleic acid is newly configured to bind to a newsite-directed polypeptide. The new site-directed polypeptide cancomprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50% or moreamino acid sequence identity to a wild-type site-directed polypeptide.The new site-directed polypeptide can comprise at most about 5, 10, 15,20, 25, 30, 35, 40, 45 or 50% or more amino acid sequence identity to awild-type site-directed polypeptide. The new site-directed polypeptidecan be a homologue of Cas9. The new site-directed polypeptide can be anorthologue of Cas9. The new site-directed polypeptide can be a chimeraof two different site-directed polypeptides. The new site-directedpolypeptide can comprise a mutation as disclosed herein.

An engineered nucleic acid-targeting nucleic acid comprising a modifiedbulge can be engineered to bind to a site-directed polypeptide with alower or higher dissociation constant than a nucleic acid-targetingnucleic acid that does not comprise a modified bulge (e.g., wild-typenucleic acid-targeting nucleic acid). An engineered nucleicacid-targeting nucleic acid comprising a modified bulge can beengineered to bind to a site-directed polypeptide with a dissociationconstant at least about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450,500, 550 or 600% or more lower or higher than a nucleic acid-targetingnucleic acid that does not comprise a modified bulge. An engineerednucleic acid-targeting nucleic acid comprising a modified bulge can beengineered to bind to a site-directed polypeptide with a dissociationconstant at most about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450,500, 550 or 600% or more lower or higher than a nucleic acid-targetingnucleic acid that does not comprise a modified bulge. An engineerednucleic acid-targeting nucleic acid comprising a modified bulge can beengineered to bind to a site-directed polypeptide with a dissociationconstant at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold,20-fold, 30-fold, 40-fold, or 50-fold or more lower or higher than anucleic acid-targeting nucleic acid that does not comprise a modifiedbulge. An engineered nucleic acid-targeting nucleic acid comprising amodified bulge can be engineered to bind to a site-directed polypeptidewith a dissociation constant at most about 1-fold, 2-fold, 3-fold,4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold or morelower or higher than a nucleic acid-targeting nucleic acid that does notcomprise a modified bulge.

Methods

The disclosure provides for methods for engineering nucleicacid-targeting nucleic acids. The methods can comprise modifying anucleic acid-targeting nucleic acid. The modifying can compriseinserting, deleting, substituting, and mutating the nucleotides in thenucleic acid-targeting nucleic acid. The modifying can comprisemodifying the nucleic acid-targeting nucleic acid such that the nucleicacid-targeting nucleic acid can bind to new protospacer adjacent motifsand/or new site-directed polypeptides as compared to a wild-type nucleicacid-targeting nucleic acid. The method can be performed using any ofthe site-directed polypeptides, nucleic acid-targeting nucleic acids,and complexes of site-directed polypeptides and nucleic acid-targetingnucleic acids as described herein.

An engineered nucleic acid-targeting nucleic acid can be used to cleavea target nucleic acid. An engineered nucleic acid-targeting nucleic acidcan be introduced into cells with a site-directed polypeptide, therebyforming a complex. The complex can hybridize to a target nucleic acid,wherein the target nucleic acid comprises a protospacer adjacent motif.The site-directed polypeptide of the complex can cleave the targetnucleic acid.

Complementary portions of the nucleic acid sequences of pre-CRISPRnucleic acid and tracr nucleic acid sequences from Streptococcuspyogenes SF370 are shown in FIG. 20.

FIG. 21 depicts exemplary structure of the duplex (e.g., hairpin)comprising the minimum CRISPR repeat and the minimum tracrRNA sequenceand a portion of the 3′ tracrRNA sequence. The duplex comprises a bulgeregion.

Table 4 contains the sequences of DNA templates used to synthesize thesingle guide nucleic acid-targeting nucleic acids of the disclosure.

TABLE 4 DNA templates of the guide nucleic acids of the disclosureDuplex variants group 1 TEMP3-AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC FLTATGCTGTTTTGGAAACAAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v2AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGAAAAAGAGCTAGAAATAGCAAGTTTTTTTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v3AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGATATAGAGCTAGAAATAGCAAGTTATATTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v4AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGGATGAAAATCCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v5AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGAAAATGAGGATGAAAATCCAAGTATTTTTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v6AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGATTATGAGGATGAAAATCCAAGTATAATTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v7AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTAATTGAGGATGAAAATCCAAGTAATTATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v8AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGAAAATCAAGTGATGAAAATCGAGATTTTTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v9AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGAAAATGAAGGATGAAAATCCAGTATTTTTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v10AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGATTTAGAGCTAGAAATAGCAAGTTAAATTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v11AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTCTCAGAGCTAGAAATAGCAAGTTGAGATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v12AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTCCCAGAGCTAGAAATAGCAAGTTGGGATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v13AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGACTCAGAAATCAGAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT Duplex variants group 2 SGR-v14AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTAGAAATAGCTCTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCAC CGAGTCGGTGCTTTTTTTSGR-v15 AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGGAAACTCTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC GGTGCTTTTTTTSGR-v16 AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAAATAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCT TTTTTT SGR-v17AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATATTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v18AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATATTTTAGAGCTAGAAATAGCAAGTTAAAACAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v19AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGACGATAGAACGGAAACGTTGGACATCGTTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v20AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGACGATGAGACGGAAACGTCAAGTATCGTTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v21AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAAGACTAGAAATAGTGGACTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v22AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATCGTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v23AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTGGTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v24AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTGCGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v25AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGTGAGAAATAGCAAGTTCACATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v26AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTAGAAATAGCAAGTTACACTAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v27AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTAGAAATAGCAAGTTAACAGAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v28AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAACTGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v29AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTAGAAATAGCAAGTTAAAATGCTAGTCCGTTATCAACTTGAAAAAGTGGCACC GAGTCGGTGCTTTTTTTTracr-Variant Group SGR-v30AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTAGAAATAGCAAGTTAAAATGGAACTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v31AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTAGAAATAGCAAGTTAAAATTTCGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v32AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGCGAAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v33AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTTCACCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v34AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTGGCTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v35AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGAATTCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v36AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTAAGTACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v37AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCATGATGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v38-AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC MMOTAGAAATAGCAAGTTAAAATAAGAATGATACATCACAAAAAAAAGGCTTTATGCCGTAACTACTACTTATTTTCAAAATAAGTAGTTTTTTTT SGR-v39-AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC ST2TAGAAATAGCAAGTTAAAATAAGGCTTCATGCCGAAATCAACACCCTGTCATTTTATGGCAGGGTGTTTTCGTTATTTTTTT SGR-v40-AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC CJTAGAAATAGCAAGTTAAAATAAAGAGTTTGCGGGACTCTGCGGGGTTACAATCCCCTAAAACCGCTTTTTTT SGR-v41-AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGC NMTAGAAATAGCAAGTTAAAATAAGGCCGTCTGAAAAGATGTGCCGCAACGCTCTGCCCCTTAAAGCTTCTGCTTTAAGGGGCATTTTTT Csy4-tag-group SGR-v42AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTATACTGCCGTA TAGGCAGAGATTTTTTSGR-v43  AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTATACTGCCGTATAGGCAGAGAAATGGACTCGGAATACTGCCGTATAGGCAGAGATTTTTT SGR-v44AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTATCACTGCCGTATAGGCAGTGATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT SGR-v45AGTAATAATACGACTCACTATAGGGGGCCACTAGGGACAGGATGTTTTAGAGCTATCACTGCCGTATAGGCAGTGATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTAATGGACTCGATACTGCCGTAT AGGCAGAGATTTTTT

Table 2 shows the RNA sequences of the DNA templates in Table 1.

TABLE 2 RNA sequences of single guide nucleic acid-targetingnucleic acids of the disclosure Caribou Single guide P/Nnucleic acid-targeting nucleic acid sequence Duplex variants group 1TEMP3- GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAUGCUGUUUUGGAAACA FLAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU SGR-v2GGGGCCACUAGGGACAGGAUGAAAAAGAGCUAGAAAUAGCAAGUUUUUUUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v3GGGGCCACUAGGGACAGGAUGAUAUAGAGCUAGAAAUAGCAAGUUAUAUUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v4GGGGCCACUAGGGACAGGAUGUUUUAGAGGAUGAAAAUCCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v5GGGGCCACUAGGGACAGGAUGAAAAUGAGGAUGAAAAUCCAAGUAUUUUUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v6GGGGCCACUAGGGACAGGAUGAUUAUGAGGAUGAAAAUCCAAGUAUAAUUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v7GGGGCCACUAGGGACAGGAUGUAAUUGAGGAUGAAAAUCCAAGUAAUUAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v8GGGGCCACUAGGGACAGGAUGAAAAUCAAGUGAUGAAAAUCGAGAUUUUUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v9GGGGCCACUAGGGACAGGAUGAAAAUGAAGGAUGAAAAUCCAGUAUUUUUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v10GGGGCCACUAGGGACAGGAUGAUUUAGAGCUAGAAAUAGCAAGUUAAAUUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v11GGGGCCACUAGGGACAGGAUGUCUCAGAGCUAGAAAUAGCAAGUUGAGAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v12GGGGCCACUAGGGACAGGAUGUCCCAGAGCUAGAAAUAGCAAGUUGGGAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v13GGGGCCACUAGGGACAGGAUGUUUUAGACUCAGAAAUCAGAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUUDuplex variants group 2 SGR-v14GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCUCUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU UUUUU SGR-v15GGGGCCACUAGGGACAGGAUGUUUUAGAGGAAACUCUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU SGR-v16GGGGCCACUAGGGACAGGAUGUUUUAGAAAUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU SGR-v17GGGGCCACUAGGGACAGGAUAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v18GGGGCCACUAGGGACAGGAUAUUUUAGAGCUAGAAAUAGCAAGUUAAAACAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v19GGGGCCACUAGGGACAGGAUGACGAUAGAACGGAAACGUUGGACAUCGUUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v20GGGGCCACUAGGGACAGGAUGACGAUGAGACGGAAACGUCAAGUAUCGUUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v21GGGGCCACUAGGGACAGGAUGUUUUAAGACUAGAAAUAGUGGACUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v22GGGGCCACUAGGGACAGGAUCGUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v23GGGGCCACUAGGGACAGGAUGUGGUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v24GGGGCCACUAGGGACAGGAUGUUUGCGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v25GGGGCCACUAGGGACAGGAUGUUUUAGAGUGAGAAAUAGCAAGUUCACAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v26GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUACACUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v27GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAACAGAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v28GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAACUGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCUUUUUUU SGR-v29GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU UUUUTracr-Variant Group SGR-v30GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUGGAACUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v31GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUUUCGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v32GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGCGAAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v33GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUUCACCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v34GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUGGCUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v35GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGAAUUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v36GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAAGUACUUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v37GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAUGAUGAAAAAGUGGCACCGAGUCGGUGC UUUUUUU SGR-v38-GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA MMOAUAAGAAUGAUACAUCACAAAAAAAAGGCUUUAUGCCGUAACUACUACUUAUUUUCAAAAUAAGUAGUUUUUUU SGR-v39-GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA ST2AUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG UUUUCGUUAUUUUUUUSGR-v40- GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA CJAUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCU UUUUUU SGR-v41-GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAA NMAUAAGGCCGUCUGAAAAGAUGUGCCGCAACGCUCUGCCCCUUAAAGCU UCUGCUUUAAGGGGCAUUUUUUCsy4-tag-group SGR-v42  GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUAUACUGCCGUAUAGGCAGAGAUUU UUU SGR-v43GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUAUACUGCCGUAUAGGCAGAGAAAUGGACUCGGAAUACUGCCGUAUAGGCAGAGAUUUUUU SGR-v44GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAUCACUGCCGUAUAGGCAGUGAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU SGR-v45GGGGCCACUAGGGACAGGAUGUUUUAGAGCUAUCACUGCCGUAUAGGCAGUGAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUAAUGGACUCGAUACUGCCGUAUAGGCAGAG AUUUUUU

Table 5 indicates the activity and purpose of additional nucleicacid-targeting nucleic acid variants. + refers to active, − refers toinactive. The experimental data of these variants is shown in FIG. 37.

Nucleic acid-targeting nucleic acid variants and their activity namedCB ### Actvity Purpose Spacer sgBackbone SGR- - Linker GGGGCCGTTTTAGAGCTAGAAATAGC v80 length ACTAGG AAGTTAAAATAACTCGGCTA GACAGGTCCGTTATCAACTTGAAAA GAT AGTGGCACCGAGTCGGTGCT SGR- - Linker GGGGCCGTTTTAGAGCTAGAAATAGC v81 length ACTAGG AAGTTAAAATAACTCTGGCT GACAGAGTCCGTTATCAACTTGAAA GAT AAGTGGCACCGAGTCGGTGC T SGR- - Linker GGGGCCGTTTTAGAGCTAGAAATAGC v82 length ACTAGG AAGTTAAAATAACTCTCTGG GACAGCTAGTCCGTTATCAACTTGA GAT AAAAGTGGCACCGAGTCGGT GCT SGR- ++ Control GGGGCCGTTTTAGAGCTAGAAATAGC v83 ACTAGG AAGTTAAAATAAGGCTAGTC GACAGCGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR- ++ Minimal GGGGCCGTTTTAGAGGAAACAAGTTA v84 sgRNA ACTAGG AAATAAGGCTAGTCCGTTAT GACAGCAACTTGAAAAAGTGGCACC GAT GAGTCGGTGCT SGR- ++ Minimal GGGGCCGTTTTAGAGACAAGTTAAAA v85 sgRNA ACTAGG TAAGGCTAGTCCGTTATCAA GACAGCTTGAAAAAGTGGCACCGAG GAT TCGGTGCT SGR- ++ Minimal GGGGCCGTTTTAGGAGAAACTTTAAA v86 sgRNA ACTAGG ATAAGGCTAGTCCGTTATCA GACAGACTTGAAAAAGTGGCACCGA GAT GTCGGTGCT SGR- - Minimal GGGGCCGTTTTATCGAAATCTAAAAT v87 sgRNA ACTAGG AAGGCTAGTCCGTTATCAAC GACAGTTGAAAAAGTGGCACCGAGT GAT CGGTGCT SGR- - Minimal GGGGCCGTTTTACTTCGGTAAAATAA v88 sgRNA ACTAGG GGCTAGTCCGTTATCAACTT GACAGGAAAAAGTGGCACCGAGTCG GAT GTGCT SGR- ++ Minimal GGGGCCGTTTTAGATACTTAAAATAA v89 sgRNA ACTAGG GGCTAGTCCGTTATCAACTT GACAGGAAAAAGTGGCACCGAGTCG GAT GTGCT SGR- - Minimal GGGGCCGTTTTATGAAACTAAAATAA v90 sgRNA ACTAGG GGCTAGTCCGTTATCAACTT GACAGGAAAAAGTGGCACCGAGTCG GAT GTGCT SGR- - Minimal GGGGCCGTTTCTTCGGAAATAAGGCT v91 sgRNA ACTAGG AGTCCGTTATCAACTTGAAA GACAGAAGTGGCACCGAGTCGGTGC GAT T SGR- ++ Change GGGGCC GTTTTAGGCTAGAAATAGCAv92 bulge ACTAGG AGTTAAAATAAGGCTAGTCC angle GACAG GTTATCAACTTGAAAAAGTGGAT GCACCGAGTCGGTGCT SGR- ++ Change GGGGCC GTTTTAGCTAGAAATAGCAA v93bulge ACTAGG GTTAAAATAAGGCTAGTCCG angle GACAG TTATCAACTTGAAAAAGTGG GATCACCGAGTCGGTGCT SGR- ++ Change GGGGCC GTTTTACTAGAAATAGCAAG v94 bulgeACTAGG TTAAAATAAGGCTAGTCCGT angle GACAG TATCAACTTGAAAAAGTGGC GATACCGAGTCGGTGCT SGR- - Change GGGGCC GTTTTAGAGCTAGAAATAGC v95 bulgeACTAGG AGTTAAAATAAGGCTAGTCC angle GACAG GTTATCAACTTGAAAAAGTG GATGCACCGAGTCGGTGCT SGR- - Change GGGGCC GTTTTAGAGCTAGAAATAGA v96 bulgeACTAGG GTTAAAATAAGGCTAGTCCG angle GACAG TTATCAACTTGAAAAAGTGG GATCACCGAGTCGGTGCT SGR- ++ Change GGGGCC GTTTTAGAGCTAGAAATAAG v97 bulgeACTAGG TTAAAATAAGGCTAGTCCGT angle GACAG TATCAACTTGAAAAAGTGGC GATACCGAGTCGGTGCT SGR- - Change GGGGCC GTTTTAGAGCTAGAAATAGT v98 bulgeACTAGG TAAAATAAGGCTAGTCCGTT angle GACAG ATCAACTTGAAAAAGTGGCA GATCCGAGTCGGTGCT SGR- - Control GGGGCC GTTTTAGAGCTAGAAATAGC v99 ACTAGGAAGTTAAAATAAGGCTAGTC GACAG CGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCTSGR- dCB154 - nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC v124 airpin ACTAGGAAGTTAAAATAACCCTAGTC variant GACAG CGTTATCAACTTGAAAAAGT GATGGCACCGAGTCGGTGCT SGR- dCB155 +/- nexus/h GGGGCC GTTTTAGAGCTAGAAATAGCv125 airpin ACTAGG AAGTTAAAATAACCCTAGTG variant GACAGGGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR- dCB156 + nexus/h GGGGCCGTTTTAGAGCTAGAAATAGC v126 airpin ACTAGG AAGTTAAAATAAGGCTAGTG variantGACAG GGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR- dCB157 ++ nexus/hGGGGCC GTTTTAGAGCTAGAAATAGC v46 airpin ACTAGG AAGTTAAAATAAGGCTAGTAvariant GACAG AGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR- dCB158 ++nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC v47 airpin ACTAGGAAGTTAAAATAAGGCTAGTT variant GACAG TGTTATCAACTTGAAAAAGT GATGGCACCGAGTCGGTGCT SGR- dCB159 ++ nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC v48airpin ACTAGG AAGTTAAAATAAGACTAGTT variant GACAG CGTTATCAACTTGAAAAAGTGAT GGCACCGAGTCGGTGCT SGR- dCB160 - nexus/h GGGGCC GTTTTAGAGCTAGAAATAGCv49 airpin ACTAGG AAGTTAAAATAAGGCTAGGA variant GACAG AACTAGCCTTCTCAA GATSGR- dCB161 - nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC v50 airpin ACTAGGAAGTTAAAATAAGGCTAGGA variant GACAG AACTAGCCCTCAATA GAT SGR- dCB162 +/-nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC v51 airpin ACTAGGAAGTTAAAATAAGGCTAGTC variant GACAG CGTAGAAAATGCC GAT SGR- dCB163 +nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC v52 airpin ACTAGGAAGTTAAAATAAGGCTAGTC variant GACAG CGTAGAAATACGG GAT SGR- dCB164 +nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC v53 airpin ACTAGGAAGTTAAAATAAGGCTAGTC variant GACAG CGTAGAAATACTTAT GAT SGR- dCB165 +nexus/h GGGGCC GTTTTAGAGCTAGAAATAGC v54 airpin ACTAGGAAGTTAAAATAAGGCTAGTC variant GACAG CGTTATTATTAGGGGGTTA GAT SGR- dCB166 -nexus GGGGCC GTTTTAGAGCTAGAAATAGC v55 trunc- ACTAGG AAGTTAAAATAAGG ationGACAG GAT SGR- dCB167 - nexus GGGGCC GTTTTAGAGCTAGAAATAGC v56 trunc-ACTAGG AAGTTAAAATAAGGCTAG ation GACAG GAT SGR- dCB168 +/- nexus GGGGCCGTTTTAGAGCTAGAAATAGC v57 trunc- ACTAGG AAGTTAAAATAAGGCTAGTC ation GACAGCG GAT SGR- dCB169 +/- nexus GGGGCC GTTTTAGAGCTAGAAATAGC v58 trunc-ACTAGG AAGTTAAAATAAGGCTAGTC ation GACAG CGTTAT GAT SGR- +/- nexus GGGGCCGTTTTAGAGCTAGAAATAGC v100 stem ACTAGG AAGTTAAAATAAGGGCTAGT length GACAGCCCGTTATCAACTTGAAAAA GAT GTGGCACCGAGTCGGTGCT SGR- ++ nexus GGGGCCGTTTTAGAGCTAGAAATAGC v101 stem ACTAGG AAGTTAAAATAAGGGGCTAG length GACAGTCCCCGTTATCAACTTGAAA GAT AAGTGGCACCGAGTCGGTGC T SGR- + nexus GGGGCCGTTTTAGAGCTAGAAATAGC v102 stem ACTAGG AAGTTAAAATAAGGGGGCTA length GACAGGTCCCCCGTTATCAACTTGA GAT AAAAGTGGCACCGAGTCGGT GCT SGR- ++ nexus GGGGCCGTTTTAGAGCTAGAAATAGC v103 stem ACTAGG AAGTTAAAATAAAACTAGTT length GACAGTGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR- ++ nexus GGGGCCGTTTTAGAGCTAGAAATAGC v104 stem ACTAGG AAGTTAAAATAAGGACTAGT length GACAGTCCGTTATCAACTTGAAAAA GAT GTGGCACCGAGTCGGTGCT SGR- ++ Control GGGGCCGTTTTAGAGCTAGAAATAGC v105 ACTAGG AAGTTAAAATAAGGCTAGTC GACAGCGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR- +/- nexus GGGGCCGTTTTAGAGCTAGAAATAGC v106 stem ACTAGG AAGTTAAAATAAGGCTAGTC length GACAGCTGTTATCAACTTGAAAAAG GAT TGGCACCGAGTCGGTGCT SGR- ++ nexus GGGGCCGTTTTAGAGCTAGAAATAGC v107 stem ACTAGG AAGTTAAAATAAAGCTAGTC length GACAGTGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR- - nexus GGGGCCGTTTTAGAGCTAGAAATAGC v108 loop ACTAGG AAGTTAAAATAAGGTCTAGT size GACAGCCCGTTATCAACTTGAAAAA GAT GTGGCACCGAGTCGGTGCT SGR- + nexus GGGGCCGTTTTAGAGCTAGAAATAGC v109 loop ACTAGG AAGTTAAAATAAGGTACTAG size GACAGTCGCCGTTATCAACTTGAAA GAT AAGTGGCACCGAGTCGGTGC T SGR- - nexus GGGGCCGTTTTAGAGCTAGAAATAGC v110 loop ACTAGG AAGTTAAAATAAGGTATCTA size GACAGGTGCGCCGTTATCAACTTGA GAT AAAAGTGGCACCGAGTCGGT GCT SGR- ++ Control GGGGCCGTTTTAGAGCTAGAAATAGC v111 ACTAGG AAGTTAAAATAAGGCTAGTC GACAGCGTTATCAACTTGAAAAAGT GAT GGCACCGAGTCGGTGCT SGR- - Tracr GGGGCCGTTTTAGAGCTAGAAATAGC v112 hybrid- ACTAGG AAGTTAAAATCAAACAAAGC LRH GACAGTTCAGCTGAGTTTCAATTTCT GAT GGCCCATGTTGGGCACATAC ATATGCCACCGAG SGR- ++Tracr GGGGCC GTTTTAGAGCTAGAAATAGC v113 hybrid- ACTAGGAAGTTAAAATAAGGCAGTGA SM159 GACAG TTTTTAATCCAGTCCGTACAC GATAACTTGAAAAAGTGCGCACC GATTCGGTGCTTTTTT SGR- ++ Tracr GGGGCCGTTTTAGAGCTAGAAATAGC v114 hybrid- ACTAGG AAGTTAAAATAAGGCTTAGA ST3 GACAGCCGTACTCAACTTGAAAAGG GAT TGGCACCGATTCGGTGTTTTT TTT SGR- ++ Tracr GGGGCCGTTTTAGAGCTAGAAATAGC v115 hybrid- ACTAGG AAGTTAAAATCAAAGCGCTT LBU GACAGTGCGCGGAGTTTCAACTTTT GAT SGR- - Major GGGGCC GAGAATCTCCTAGAAATAGC v120variant ACTAGG TCTTATTCTTAAGGGATCACC GACAG GAATACAACTTGAAAAAGTG GATGCACCGAGTCGGTGCT SGR- ++ Major GGGGCC GACGATGAGACGGAAACGTC v121 variantACTAGG AAGTATCGTTAAGGGATCAC GACAG CGAATACAACTTGAAAAAGT GATGGCACCGAGTCGGTGCT SGR- - Major GGGGCC AACGATGAGACGGAAACGTC v122 variantACTAGG AAGTATCGTCAAGGGATCAC GACAG CCAATACAACTTGAAAAAGT GATGGCACCGAGTCGGTGCT SGR- - Major GGGGCC AACGGTGAGGTGGAAACACC v123 variantACTAGG AAGTACCGTCAAGGTAGCAC GACAG CCGACAAGTC GAT

Methods for the Generation of Tagged Cell Lines Using a NucleicAcid-Targeting Nucleic Acid

The methods of the disclosure provide for tagging a cell with a donorpolynucleotide, wherein the donor polynucleotide can divide and/ordifferentiate, and the donor polynucleotide can be transmitted to eachdaughter cell during cell division. The method can be performed usingany of the site-directed polypeptides, nucleic acid-targeting nucleicacids, and complexes of site-directed polypeptides and nucleicacid-targeting nucleic acids as described herein.

A tagged cell can be generated by contacting the cell with a donorpolynucleotide, and a complex comprising a site-directed polypeptide anda nucleic acid-targeting nucleic acid. The donor polynucleotide can beinserted into the cleaved target nucleic acid, thereby generating atagged cell. The tagged cell can be propagated such as in a cell line,or to produce a propagated population of cells.

A donor polynucleotide can be introduced into the cut site by use of adonor cassette for homologous recombination that comprises endshomologous to sequences on either side of the double-strand break. Thedonor polynucleotide can comprise an additional sequence between the twoends. The additional sequence can be a nucleic acid sequence. Theadditional sequence can encode for a gene. The additional sequence canencode for a non-coding nucleic acid element.

The donor polynucleotide (e.g., the additional sequence of a donorpolynucleotide between two homologous ends) can comprise a marker. Amarker can comprise a visualization marker (e.g., a fluorescent markersuch as GFP). A marker can comprise a random polynucleotide sequence(e.g., such as a random hexamer sequence). A marker can be a barcode.

NHEJ can introduce unique sequence signature at each cut site. Therepair mechanism can result in the introduction of insertions (e.g.,insertion of a donor polynucleotide), deletions or mutations into a cutsite. A cell that undergoes NHEJ to repair a double-strand break cancomprise a unique sequence after repair has taken place (e.g., a uniquesequence can be inserted into the double-strand break). If more than onesite is cut within a cell, repair can introduce the donor polynucleotideat each site, thereby adding sequence diversity to that cell. Therepaired site can provide a unique barcode sequence to the cell that canbe preserved during cell division and passed on to all progeny of themodified cell. A donor polynucleotide can be inserted into at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more sites (e.g, cleaved targetnucleic acids). A donor polynucleotide can be inserted into at mostabout 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more sites (e.g, cleaved targetnucleic acids).

Homologous recombination (HR) can be used to introduce barcode sequencesinto a cell and/or a cell population (e.g., a human cell, a mammaliancell, a yeast, a fungi, a protozoa, an archaea). A library of donorplasmids (e.g., comprising the donor polynucleotide) can be preparedwith randomized sequences in the donor cassette. The library can be madefrom oligonucleotides, a piece of double-stranded DNA, a plasmid, and/ora minicircle.

Donor polynucleotide sequences can be introduced into the genomes ofindividual cells for the purpose of tracking cell lineage. Sites can bechosen for modification in silent or “safe-harbor” regions of thegenome, distant from genes and regulatory elements, to minimizepotentially deleterious effects on cellular function. Sites withinfunctional genetic elements can also be used to track cell fate.

For example, donor polynucleotides can be introduced into stem celland/or stem cell populations. The methods of the disclosure can be usedfor tracking cell lineage development in animal models. For example,cell fate development and/or differentiation in hematopoiesis can betracked using the methods of the disclosure. The methods of thedisclosure can be used for therapeutic cell engineering-based therapies.For example, a cell can be tagged with a donor polynucleotide encoding atherapeutic protein. The cell can be propagated. The propagated cell canbe introduced into a subject. As another example, a differentiated cellcan be removed from a subject. The differentiated cell can be taggedwith two markers: one expressed when the cell is differentiated, oneexpressed when the cell is de-differentiated. Identifying the markerscan be useful in determining differentiation events. In another example,a differentiated cell can be obtained from a subject. The differentiatedcell can be de-differentiated into a pluripotent cell. The pluripotentcell can be tagged with a donor polynucleotide encoding a therapeuticprotein. The cell can be re-differentiated into a new cell type whileexpressing the therapeutic protein, thereby creating a patient-specifictherapeutic cell. Tagged cells can divide and differentiate, and themodification(s) to their genome can be transmitted to each daughter cellduring cell division.

In some instances, two cells can be tagged with two different donorpolynucleotide markers. The two cells can be combined. The combinedmixture can be assayed simultaneously. The donor polynucleotides canallow the multiplex analysis of the two cells because the donorpolynucleotide can be used to distinguish the two cells.

A cell population can be chosen for introducing double-stranded breaks,or generating cellular signatures. Cells may be purified or selected.For example, a population of hematopoetic stem cells (CD45positive) maybe selected by FACS or magnetic bead purification. Bone marrow may betreated ex vivo with the nuclease. Cells may be targeted in vivo by theuse of viruses with a particular tropism. Cells may be selected by usingviruses engineered to target cells bearing a particular receptor.

Tagged cells can be analyzed by high-throughput sequencing either at thepopulation level or at the single-cell level. At the population level, acollection of cells can be lysed. The genomic DNA can be extracted. PCRprimers can be designed to amplify the genomic region that has beenmodified by the nuclease. Sequences can be enriched by hybridization. Asequence library can be prepared from the genomic DNA and enriched. Theregion of interest can be enriched, and a sequence library can beprepared. A sequence library can be prepared simultaneously duringenrichment using primers comprising appropriate sequence tags to be usedwith nucleic acid sequencing technologies. If the double-stranded breakis made within a region that can be transcribed, RNA can be used toprepare sequence libraries.

Once nucleic acid sequence data has been obtained, the sequences can beanalyzed to determine the clonal structure. This can be carried out bygathering common sequences together and counting those sequences.

Cells can be sub-selected by sorting schemes based on cell surfacemarkers using flow cytometry or affinity purification methods. Cellsurface markers can be used to define cell states, and by comparing cellstates with clonal structure, the fate of modified cell populations canbe determined.

At the single-cell level, cells can be isolated. PCR products can begenerated from each individual cell. This can be achieved in microwellarrays, microfluidic devices, and/or emulsions. Where more than onegenomic modification is carried out per cell, PCR products can becoupled together, either physically, or chemically, to ensure theirrelationship to the parent cell.

Methods for Quantifying Genome-Editing Events

For RNA-dependent nucleases, such as Cas9, the nucleic acid recognitionfunctionality and nuclease activities can be linked. In some instances,nucleic acid recognition functionality and nuclease activities may notbe linked. The nuclease sites can be located within the specificsequence recognized by the nuclease.

Non-Homologous End-Joining can be an imperfect repair process that canresult in the insertion of multiple bases at the site of the doublestranded break. NHEJ can result in the introduction of insertions,deletions and/or mutations into a cut site. NHEJ can significantlydisrupt the original sequence. The disruption of the native sequence asa consequence of repair mechanisms can be used to assess the efficiencyof genome editing approaches.

Homologous recombination can enable more complete repair of the targetnucleic acid break by exchanging nucleotide sequences between similar oridentical molecules of nucleic acid. An additional sequence can beintroduced into the target nucleic acid at the cut site by use of adonor cassette (e.g., donor polynucleotide) that comprises endshomologous to sequences either side of the double-strand break andadditional sequence between the two ends.

This disclosure describes an approach for assessing double-strandedbreak activity and NHEJ-mediated insertions/deletions introduced bynucleic acid-dependent nucleases, such as Cas9. The method takesadvantage of the fact that the sites in a target nucleic acid recognizedby Cas9 during the initial nuclease recognition and nucleic acidcleavage activity can be destroyed during the NHEJ process, either bythe introduction of insertions or deletions.

In some instances, the method provides for the design of a nucleicacid-targeting nucleic acid to target a site of interest in a targetnucleic acid (e.g., genome). A nucleic acid template encoding thenucleic acid-targeting nucleic acid can be designed with a promotersequence appended at the 5′ end of the nucleic acid-targeting nucleicacid to enable in vitro synthesis of the nucleic acid-targeting nucleicacid.

Primers can be designed at positions that flank the cleavage site. Thecleavage site (and/or nucleic acid regions around the cleavage site) canbe amplified (e.g., from genomic nucleic acid), thereby generating aproduct (e.g., amplified PCR product). The product (e.g., amplified PCRproduct) can be at least about 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 1100, 1200 or more bases in length. The product (e.g.,amplified PCR product) can be at most about 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1100, 1200 or more bases in length. Theproduct (e.g., amplified PCR product) can be about 200-600 base pairs inlength.

The products can be purified. The products can be incubated with anRNA-dependent nuclease (e.g. Cas9) and the nucleic acid-targetingnucleic acid. Those molecules that have been amplified from genomicnucleic acid that have not be modified by NHEJ can comprise the correctsequence that can be recognized and cleaved by Cas9. The molecules thathave been amplified from genomic nucleic acid that has been modified byNHEJ may not comprise sites that can be recognized and/or cut by Cas9.

The digested products can then be analyzed by methods such as gelelectrophoresis, capillary electrophoresis, high-throughput sequencingand/or quantitative PCR (e.g., qPCR). In the case of gelelectrophoresis, a gel can be imaged. Once a gel has been imaged, thepercentage of cells modified by NHEJ can be estimated by measuring theintensity of bands corresponding to digested products, and comparing tothe intensity of bands corresponding to undigested products.

Methods for Delivering Donor Polynucleotide to a Double-Stranded Breakfor Insertion into the Double-Stranded Break

This disclosure describes methods for bringing a donor polynucleotideinto close proximity to a site-directed target nucleic acid break toenhance insertion (e.g., homologous recombination) of the donorpolynucleotide into the site of the double-stranded break. The methodcan be performed using any of the site-directed polypeptides, nucleicacid-targeting nucleic acids, and complexes of site-directedpolypeptides and nucleic acid-targeting nucleic acids as describedherein.

In some instances, the methods of the disclosure provide for forbringing a donor polynucleotide in close proximity to the site of adouble-stranded break in a target nucleic acid, by binding it to thenuclease that generates the double-stranded break (e.g., Cas9).

A complex comprising a site-directed polypeptide, a nucleicacid-targeting nucleic acid, and a donor polynucleotide can be deliveredto a target nucleic acid. FIG. 30 illustrates exemplary methods forbringing a donor polynucleotide into proximity to the site of adouble-stranded break in a target nucleic acid. For example, a nucleicacid-targeting nucleic acid can comprise a 3′ hybridizing extensionsequence, which can be part of a tracrRNA extension sequence (shown inthe light dotted line attached to the nucleic acid-targeting nucleicacid). A 3′ hybridizing extension sequence can be a non-native sequence.FIG. 30A illustrates that the tracrRNA extension sequence at the 3′ endof the nucleic acid-targeting nucleic acid can include a sequence thatcan hybridize to an end of the donor polynucleotide (e.g., the 3′ end)(the donor polynucleotide is shown in bold thicker dashed line). The 3′hybridizing extension sequence can be at least about 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 or more nucleotides in length. The 3′ hybridizingextension sequence can be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10or more nucleotides in length. The 3′ hybridizing sequence can hybridizeto at least about 1, 2, 3, 4, 5, 5, 6, 7, 8, 9, or 10 or morenucleotides of the donor polynucleotide. The 3′ hybridizing sequence canhybridize to at most about 1, 2, 3, 4, 5, 5, 6, 7, 8, 9, or 10 or morenucleotides of the donor polynucleotide. The 3′ hybridizing sequence canhybridize to the donor polynucleotide with at least 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or more mismatches. The 3′ hybridizing sequence canhybridize to the donor polynucleotide with at most 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or more mismatches.

The 3′ hybridizing extension can hybridize to the 3′ end of the donorpolynucleotide. The 3′ hybridizing extension can hybridize to at leastthe 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 3′ most nucleotides of thedonor polynucleotide. The 3′ hybridizing extension can hybridize to atmost the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 3′ most nucleotides ofthe donor polynucleotide.

As depicted in FIG. 30B, the tracr nucleic acid extension at the 3′ endof the nucleic acid-targeting nucleic acid can include a sequence thatcan hybridize to the 5′ end of the donor DNA. The 3′ hybridizingextension can hybridize to at least the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10or more 5′ most nucleotides of the donor polynucleotide. The 3′hybridizing extension can hybridize to at most the 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or more 5′ most nucleotides of the donor polynucleotide.

The tracr nucleic acid extension at the 3′ end of the nucleicacid-targeting nucleic acid can include a sequence that can hybridize toa region between the 3′ end and 5′ end of the donor polynucleotide, asshown in FIG. 30C. The 3′ hybridizing extension can hybridize to atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides between the3′ and 5′ end of the donor polynucleotide. The 3′ hybridizing extensioncan hybridize to at most the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or morenucleotides between the 3′ and 5′ end of the donor polynucleotide.

The tracr nucleic acid extension at the 3′ end of the nucleicacid-targeting nucleic acid can include a sequence that can hybridizealong the full length of the donor polynucleotide, as shown in FIG. 30D.The nucleic acid-targeting nucleic acid can hybridize along at leastabout 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the donorpolynucleotide. The nucleic acid-targeting nucleic acid can hybridizealong at most about 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the donorpolynucleotide. The 3′ hybridizing extension sequence can hybridizealong the full length of the donor polynucleotide with at least about 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 or more mismatches. The 3′ hybridizingextension sequence can hybridize along the full length of the donorpolynucleotide with at most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ormore mismatches.

The tracr nucleic acid extension at the 3′ end of the nucleicacid-targeting nucleic acid (e.g., 3′ hybridizing extension) cancomprise a sequence that can be used as a template and converted by, forexample, a reverse transcriptase to generate hybrid nucleic acid (e.g.,the resulting nucleic acid is an RNA-DNA hybrid, wherein the newlytranscribed nucleic acid can be DNA), as shown in FIG. 30E. Exemplaryreverse transcriptases include SuperScript, ThermoScript, HIV reversetranscriptase, and MMLV reverse transcriptase. The reverse transcriptasecan extend the donor polynucleotide sequence from the 3′ hybridizingextension template.

The tracr nucleic acid extension at the 3′ end of the nucleicacid-targeting nucleic acid can incorporate a nucleic acid sequence thatcan binds an RNA binding protein (RBP). The RNA-binding protein can befused to a DNA binding protein (DBP), as shown in FIG. 30F. TheDNA-binding protein can bind to the donor polynucleotide.

The sequences used to bring the donor polynucleotide into closeproximity with a double-stranded break can be appended to the 5′ end ofthe nucleic acid-targeting nucleic acid (e.g., the spacer extension).The sequences sequences used to bring the donor polynucleotide intoclose proximity with a double-stranded break can be appended to both the5′ end and the 3′ end of the nucleic acid-targeting nucleic acid.

The nuclease used in the methods of the disclosure (e.g., Cas9) cancomprise nickase activity in which the nuclease can introducesingle-stranded breaks in a target nucleic acid. Pairs of nucleases withnickase activity can be targeted to regions in close proximity to eachother. A first nuclease can bind to a first nucleic acid-targetingnucleic acid that can interact with a first donor polynucleotide. Asecond nuclease can bind to a second nucleic acid-targeting nucleic acidthat can interact with a second donor polynucleotide. The first andsecond donor polynucleotides can be designed to hybridize with eachother to make a double-stranded donor polynucleotide. Two separate donorpolynucleotides can be brought to the nuclease site.

In some embodiments, the donor polynucleotide can be single stranded. Insome embodiments, the donor polynucleotide can be double stranded. Insome embodiments, the donor DNA can be a minicircle. In someembodiments, the donor polynucleotide can be a plasmid. In someembodiments, the plasmid can be supercoiled. In some embodiments, thedonor polynucleotide can be methylated. In some embodiments, the donorpolynucleotide can be unmethylated. The donor polynucleotide cancomprise a modification. Modifications can include those described hereincluding, but not limited to, biotinylation, chemical conjugate, andsynthetic nucleotides.

Methods for Cloning and Expressing a Vector Comprising a Site-DirectedPolypeptide and a Nucleic Acid-Targeting Nucleic Acid

The disclosure provides for methods for cloning an engineered nucleicacid-targeting nucleic acid into a vector (e.g., a linearized vector).The method can be performed using any of the site-directed polypeptides,nucleic acid-targeting nucleic acids, and complexes of site-directedpolypeptides and nucleic acid-targeting nucleic acids as describedherein.

A user (e.g., a scientist) can design single-stranded DNAoligonucleotides. The single-stranded DNA oligonucleotides can, whenhybridized together encode a spacer sequence to target a target nucleicacid. The single-stranded DNA oligonucleotides can be at least about 5,10, 15, 20, 25, 30 or more nucleotides in length. The single-strandedDNA oligonucleotides can be at most about 5, 10, 15, 20, 25, 30 or morenucleotides in length. The single-stranded DNA oligonucleotides can be19-20 nucleotides in length.

A single-stranded DNA oligonucleotide can be designed such that it canhybridize to a target nucleic acid (e.g., a sequence adjacent to aprotospacer adjacent motif, such as the 3′ or 5′ end of the protospaceradjacent motif). The DNA oligonucleotide can encode a sequencecorresponding to the sense or antisense strand of the target nucleicacid sequence.

The single-stranded oligonucleotides can comprise a first portion thatcan hybridize and/or is complementary to a target nucleic acid. Thesingle-stranded oligonucleotides can comprise a first portion that canhybridize and/or is complementary another single-strandedoligonucleotide. The single-stranded oligonucleotide can comprise asecond portion that can hybridize to a sequence in the linearizedvector. In other words, a pair of single-stranded oligonucleotides cancomprise a first portion that hybridizes to each other and a secondportion that comprise single-stranded overhangs, wherein the overhangscan hybridize to sticky ends in the linearized vector. In someinstances, an overhang comprises 5′-GTTTT-3′. In some instances, anoverhang comprises 5′-CGGTG-3′

The single stranded DNA nucleotides can be annealed together to generatea double-stranded oligonucleotide. The single-stranded DNA nucleotidescan be annealed together in an oligonucleotide annealing buffer (e.g.,comprising Tris-HCl, EDTA and NaCl). The double-stranded oligonucleotidecan be diluted to a working concentration (e.g., a concentrationsuitable for ligation into a linearized plasmid). The diluteddouble-stranded oligonucleotide can be ligated into a linearized vector.Ligation can be performed in a ligation buffer (e.g., comprisingTris-HCl, MgCl₂, ATP) and with a ligase (e.g., T4 DNA ligase). Thedouble-stranded oligonucleotide can be ligated into a linearized vectorat a region within the sequence encoding the nucleic acid-targetingnucleic acid. In other words, the linearized vector can be linearized ata point within the region encoding the nucleic acid-targeting nucleicacid, wherein the linearization generates sticky ends that arecomplementary to the sticky ends of the double-stranded oligonucleotide.When the double-stranded oligonucleotide is ligated into the vector, itcan generate a sequence encoding for an engineered nucleicacid-targeting nucleic acid comprising a spacer sequence correspondingto the double-stranded oligonucleotide sequence.

The ligated vector can be transformed into chemically competent cells(e.g., DH5-alpha, Top10) and selected for expression of the correctlyligated vector (e.g., by antibotic screening). The selectedtransformants can be analyzed for the presence of an insert bysequencing. Sequencing can be performed using a sequencing primer thatcan hybridize to a portion of the vector.

Correctly ligated vector can be prepared (e.g., by large scale DNApreparation, maxiprep), and purified. The vector, comprising asite-directed polypeptide, a nucleic acid-targeting nucleic acid,wherein the nucleic acid-targeting nucleic acid comprise thedouble-stranded DNA oligonucleotides can be introduced (e.g.,transfected) into a cell line of choice (e.g., mammalian cell line).

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES Example 1 Modification of a Site-Directed Polypeptide forAltered PAM Specificity

In some embodiments, the disclosure provides for a modifiedsite-directed polypeptide that is modified to alter PAM specificity. Anucleic acid encoding a modified site-directed polypeptide is introducedinto cells by transfection. The modified site-directed polypeptidecomprises an inserted HNH or RuvC nuclease domain. The modifiedsite-directed polypeptide comprises a modified highly basic patch. Anucleic acid-targeting nucleic acid that comprises a spacer that canhybridize with the target nucleic acid is also introduced into cells bytransfection. The modified site-directed polypeptide and the nucleicacid-targeting nucleic acid form a complex. The complex is guided to thetarget nucleic acid by the nucleic acid-targeting nucleic acid. Oncehybridized with the nucleic acid-targeting nucleic acid, the targetnucleic acid is cleaved by the nuclease domains of the site-directedpolypeptide. In some embodiments, the modified site-directed polypeptidebinds to the target nucleic acid with a lower Kd.

In some embodiments, a donor polynucleotide is also introduced into thecells. In some instances, the donor polynucleotide, a portion of thedonor polynucleotide, a copy of the donor polynucleotide, or a portionof a copy of the donor polynucleotide is inserted into the cleavedtarget nucleic acid.

Example 2 Modification of a Site-Directed Polypeptide for Altered TargetNucleic Acid Specificity

In some embodiments, the disclosure provides for a modifiedsite-directed polypeptide that is modified to alter target nucleic acidspecificity. A modified site-directed polypeptide is introduced intocells. The modified site-directed polypeptide comprises a modificationin the highly basic patch and/or the HNH-like domain. A nucleicacid-targeting nucleic acid that comprises a spacer that can hybridizewith the target nucleic acid is also introduced into cells. The modifiedsite-directed polypeptide and the nucleic acid-targeting nucleic acidform a complex. The complex is guided to the target nucleic acid by thenucleic acid-targeting nucleic acid. Once hybridized with the nucleicacid-targeting nucleic acid, the target nucleic acid is cleaved by thesite-directed polypeptide. In some embodiments, the modifiedsite-directed polypeptide binds to the target nucleic acid with a lowerKd.

In some embodiments, a donor polynucleotide is also introduced into thecells. In some instances, the donor polynucleotide, a portion of thedonor polynucleotide, a copy of the donor polynucleotide, or a portionof a copy of the donor polynucleotide is inserted into the cleavedtarget nucleic acid.

Example 3 Recombinant Expression of a Site-Directed Polypeptide

A recombinant DNA sequence can be assembled that encodes for a modifiedsite-directed polypeptide, and enables the expression of the modifiedsite-directed polypeptide in a host organism. The recombinant DNAsequence comprises a promoter sequence, and may additionally comprise anaffinity tag for purification, or an epitope tag. In a non-limitingexample, a plasmid comprises the recombinant DNA sequence for expressionof the modified site-directed polypeptide.

Production of Recombinant Protein.

A plasmid encoding the site directed modified polypeptide is introducedinto bacterial cells (e.g., E coli). The polypeptide is expressed inbacterial cells, and then purified from cell lysate using chromatographymethods. The activity of the modified site-directed polypeptide ismeasured using assay methods designed to determine the specificity ofthe modified polypeptide, the PAM sequence, the specificity profile ofthe site-directed polypeptide and the nucleic acid preference (forexample, DNA or RNA, or modified nucleic acids.) for the polypeptide.

Software is designed to choose sites that can be cut using the modifiedsite-directed polypeptide. Guide RNA sequences are designed to directthe activity of the site-directed polypeptide. Once designed, thesite-directed polypeptide is used to cleave nucleic acids.

Introduction of the modified site-directed polypeptide into cells

The modified peptide is introduced into cells to target nucleic acidsites. A polypeptide that retains nuclease activity is used to introducesingle-stranded or double-stranded DNA breaks into target DNA. Apolypeptide with DNA-binding but not DNA cutting activity, is used tobind double stranded DNA to a cell. This can be used to effecttranscriptional activation or repression.

Example 4 Selection of Sites for Modification within Cas9 Sequences

As described above Type II CRISPR systems containing Cas9 orthologuescan be classified into three groups (Type II-A, Type II-B, and TypeII-C) based on analysis of their CRISPR-Cas loci. Cas9 orthologueswithin these groups can be broadly defined by two clades comprisingshorter (Cas9/Csn1-type) and longer (Cas9/Csx12-type) sequences. Inaddition to these larger groups, there can be two additional families ofhomologues that comprise HNH and RuvC domains arranged with similartopology to Cas9, but with significant differences in the length andsequence of insertions between conserved sequence elements. Secondarystructure predictions and sequence alignments are used to define regionsof the polypeptide for modification. Regions that fall between secondarystructure elements or between regions of high sequence conservation areselected as candidates for insertions or deletions. Regions that havesimilarity to domains of known structure are analyzed to identifyspecific regions for inserting or deleting sequences.

FIG. 35 shows the CDD sequence alignment TIGR0185 for a small number ofdiverse Cas9 orthologs. Amino acids with a “X” below them are consideredto be similar. Amino acids with a “Y” below them can be considered to behighly conserved or identical in all sequences. Amino acids residueswithout an “X” or a “Y” are not conserved. This alignment does notinclude the C-terminal region (approximately corresponding to amino acidresidues 1100-1350) of longer Cas9 orthologs. The sequences listed inFIG. 35 are listed in Table 6.

TABLE 6 Sequences listed in FIG. 35. Genbank Accession Species gi22533915 Streptococcus agalactiae 2603V/R gi 34483507 Wolinellasuccinogenes gi 12721472 Pasteurella multocida subsp. multocida str.Pm70 gi 24377777 Streptococcus mutans UA159 gi 13622193 Streptococcuspyogenes M1 GAS gi 41815893 Treponema denticola ATCC 35405 gi 218767588Neisseria meningitidis Z2491 gi 157150687 Streptococcus gordonii str.Challis substr. CH1 gi 294660600 Mycoplasma gallisepticum str. R(low) gi218563121 Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819 gi370792169 Listeria innocua ATCC 33091

Locations for insertion of new functional domains, regions for insertionof alternative sequences, or for deletion or reduction in region sizethat modify Cas9 activity can include, but are not limited to, theregions highlighted in Table 7. The numbers represent the amino-acidsequence numbers based on the Cas9 sequence from Streptococcus pyogenesM1 GAS.

TABLE 7 Exemplary locations for modifying Cas9 gi|13622193 StreptococcusInsertion/Deletion pyogenes M1 GAS site start finish Length 1 22 42 20 297 134 37 3 170 312 142 4 350 400 50 5 426 444 18 6 455 492 37 7 528 54113 8 578 589 11 9 600 612 12 10 628 634 6 11 654 658 4 12 684 692 8 13710 727 17 14 753 756 3 15 792 794 2 16 801 804 3 17 826 834 8 18 873881 8 19 893 915 22 20 939 952 13 21 971 974 3 22 1005 1029 24 23 10961225 129 24 1105 1138 33

Once a region has been identified as a potential location for insertinga new polypeptide sequence into the protein, or deleting a region of theprotein, the DNA sequence that codes for the protein is modified toincorporate the modification.

Example 5 Sequence Enrichment of Site-Directed Polypeptide-Bound TargetNucleic Acid

The disclosure provides methods for sequence enrichment withoutamplification using site-directed polypeptides.

In some embodiments, the method will comprise a) contacting a targetnucleic acid with a complex comprising a nucleic acid-targeting nucleicacid and a site-directed polypeptide, b) cleaving the target nucleicacid c) purifying the target nucleic acid, and d) sequencing the targetnucleic acid, wherein said target nucleic acid is enriched.

In some embodiments, the site-directed polypeptide will be enzymaticallyinactive. Use of an enzymatically inactive site-directed polypeptidewill facilitate binding of the target nucleic acid to the site-directedpolypeptide complex. In some embodiments, the site-directed polypeptidewill be enzymatically active.

In some embodiments, sequence enrichment will be performed outside ofcells (e.g., cell-free sample). For example, a sample will comprisepurified genomic DNA. In some embodiments, sequence enrichment will beperformed on a cellular sample (e.g. cells, cell lysate).

In some instances, the site-directed polypeptide-target nucleic acidcomplexes will be fixed or cross-linked to form complexes. If the methodis being performed on cells, cells will be lysed. Lysis conditions willbe chosen to maintain intact protein-DNA complexes.

The nucleic acid sample will be treated to fragment the target nucleicacid before affinity purification. Fragmentation can be performedthrough physical, mechanical or enzymatic methods. Physicalfragmentation will include exposing a target polynucleotide to heat orto ultraviolet (UV) light. Mechanical disruption will be used tomechanically shear a target polynucleotide into fragments of the desiredrange. Mechanical shearing will be accomplished through a number ofmethods, including repetitive pipetting of the target polynucleotide,sonication and nebulization. Target nucleic acids will also befragmented using enzymatic methods. In some cases, enzymatic digestionwill be performed using enzymes such as using restriction enzymes.Restriction enzymes will be used to perform specific or non-specificfragmentation of target polynucleotides. The methods will use one ormore types of restriction enzymes, generally described as Type Ienzymes, Type II enzymes, and/or Type III enzymes. Type II and Type IIIenzymes recognize specific sequences of nucleotides within a doublestranded polynucleotide sequence (a “recognition sequence” or“recognition site”). Upon binding and recognition of these sequences,Type II and Type III enzymes cleave the polynucleotide sequence. In somecases, cleavage will result in a polynucleotide fragment with a portionof overhanging single stranded nucleic acid, called a “sticky end.” Inother cases, cleavage will not result in a fragment with an overhang,creating a “blunt end.” The methods may comprise use of restrictionenzymes that generate either sticky ends or blunt ends.

Once fragmented, the complexes comprising the site-directed polypeptidewill be purified by incubation with a solid support. For example, if thesite-directed polypeptide comprises a biotin tag, the solid support willbe coated with avidin or streptavidin to bind to the biotin tag.

In an alternative embodiment, once fragmented, the complexes comprisingthe site-directed polypeptide, the target nucleic acid, and/or thenucleic acid-targeting nucleic acid, will be purified by incubation witha capture agent. The capture agent will bind to the affinity tag fusedto the site-directed polypeptide. The capture agent will comprise anantibody. For example, if the affinity tag fused to the site-directedpolypeptide is a FLAG tag, then the capture agent will be ananti-FLAG-tag antibody.

In some embodiments, the capture agent will be purified with a solidsupport. For example, if the capture agent comprises a biotin tag, thesolid support will be coated with avidin or streptavidin to bind thebiotinylated capture agent.

In some embodiments, the nucleic acid-targeting nucleic acid willcomprise an affinity tag. The affinity tag will comprise a sequence thatcan bind to an endoribonuclease. In some instances, the affinity tagwill comprise a sequence that can bind to a conditionally enzymaticallyinactive endoribonuclease. The conditionally enzymatically inactiveendoribonuclease will bind, but not cleave, the affinity tag.

In some embodiments, the endoribonuclease and/or the conditionallyenzymatically inactive endoribonuclease will comprise an affinity tag.

The conditionally enzymatically inactive endoribonuclease will bepurified with a solid support. The solid support will bind to theaffinity tag of the conditionally enzymatically inactiveendoribonuclease. For example, if the conditionally enzymaticallyinactive endoribonuclease comprises a biotin tag, the solid support willbe coated with avidin or streptavidin to bind the biotinylated captureagent.

In some embodiments, the conditionally enzymatically inactiveendoribonuclease will be immobilized on any of a variety of insolublesupport.

In some embodiments of the method, two rounds of purification will beperformed. In some instances, a first round will comprise purificationwith a solid support that will bind to the affinity tag of the captureagent and a second round will comprise purification with a solid supportthat will bind to the affinity tag of the site-directed polypeptide. Insome instances, a first round will comprise purification with a solidsupport that will bind to the affinity tag of the site-directedpolypeptide and a second round will comprise purification with a solidsupport that will bind to the affinity tag of the capture agent.

In some embodiments, the methods of the disclosure will be used formultiplex sequence enrichment. In this embodiment, a plurality ofnucleic acid-targeting nucleic acids can be contacted with a nucleicacid sample, wherein each nucleic acid-targeting nucleic acid isengineered to target a different target nucleic acid (e.g., sequence ina genome) within the nucleic acid sample.

The captured complexes will comprise a target nucleic acid. The targetnucleic acid will be eluted from the site-directed polypeptide complexby standard methods including high salt washing, ethanol precipitation,boiling, gel purification, and the like.

The eluted DNA will be prepared for sequencing analysis by ligation ofone or more adaptors.

The sequencing libraries will be sequenced as described herein.Sequenced libraries will be analyzed to identify polymorphisms, diagnosea disease, determine a course of treatment for a disease, and/orgenerate antibody libraries.

Example 6 Sequence Enrichment of Target Nucleic Acid not Bound to aComplex Comprising a Site-Directed Polypeptide

In some embodiments, sequence enrichment will be performed with anenzymatically active site-directed polypeptide. In some instances, thesite-directed polypeptide will be enzymatically active. In thisinstance, the target nucleic acid will not be bound to the site-directedpolypeptide, but will be excised.

A target nucleic acid will be identified, and nucleic acid-targetingnucleic acids will be designed to direct the site-directed polypeptideto sequences that flank the target nucleic acid. The sample will beincubated with a complex comprising a designed nucleic acid-targetingnucleic acid and the site-directed polypeptide such that thesite-directed polypeptide will cleave the DNA at both ends of the targetnucleic acid. Upon cleavage of the target nucleic acid, the targetnucleic acid will be cleaved from the parent nucleic acid. The cleavedtarget nucleic acid will be purified (e.g., by gel electrophoresis,size-selective elution from beads, or other carboxylate-derivatizedbeads, or by precipitation with appropriate concentrations of salt andPEG to preferentially precipitate larger or smaller DNA).

In some embodiments, sequence enrichment will be performed outside ofcells (e.g., cell-free sample). For example, a sample will comprisepurified genomic DNA. In some embodiments, sequence enrichment will beperformed on a cellular sample (e.g. cells, cell lysate).

If the method is being performed on cells, cells will be lysed. Lysisconditions will be chosen to maintain intact protein-DNA complexes.

In some embodiments, the target nucleic acid to be sequenced will not bebound to a nucleic acid-targeting nucleic acid and/or a site-directedpolypeptide. In this embodiment, the nucleic acid bound to thesite-directed polypeptide and/or the nucleic acid-targeting nucleic acidwill be purified away. The purification of the site-directed polypeptidewill proceed as previously described herein. Briefly, the complexescomprising the site-directed polypeptide will be purified by incubationwith a solid support. For example, if the site-directed polypeptidecomprises a biotin tag, the solid support will be coated with avidin orstreptavidin to bind to the biotin tag.

In an alternative embodiment, once fragmented, the complexes comprisingthe site-directed polypeptide, the nucleic acid-targeting nucleic acid,and non-target nucleic acid, will be purified by incubation with acapture agent. The capture agent will bind to the affinity tag fused tothe site-directed polypeptide. The capture agent will comprise anantibody. For example, if the affinity tag fused to the site-directedpolypeptide is a FLAG tag, then the capture agent will be ananti-FLAG-tag antibody.

The capture agent will be purified with a solid support. For example, ifthe capture agent comprises a biotin tag, the solid support will becoated with avidin or streptavidin to bind the biotinylated captureagent.

In some embodiments, the methods of the disclosure will be used formultiplex sequence enrichment. In this embodiment, a plurality ofnucleic acid-targeting nucleic acids can be introduced into a cell,wherein each nucleic acid-targeting nucleic acid is engineered to targeta different target nucleic acid (e.g., sequence in a genome).

The captured complex will not comprise a target nucleic acid.

The target nucleic acid will comprise the nucleic acid that is not boundto the complexes comprising the site-directed polypeptide. The targetnucleic acid can be collected by standard nucleic acid purificationmethods (e.g., a commercially available PCR purification kit, an agarosegel).

The collected target nucleic acid will be prepared for sequencinganalysis (e.g., deep sequencing) by ligation of one or more adapters asdescribed herein.

Sequenced target nucleic acid will be analyzed to identifypolymorphisms, diagnose a disease, determine a course of treatment for adisease, and/or generate antibody libraries.

Example 7 Sequencing Target Nucleic Acids

The eluted target nucleic acids will be prepared for sequencinganalysis. Preparation for sequencing analysis will include thegeneration of sequencing libraries of the eluted target nucleic acid.Sequencing analysis will determine the identity and frequency ofoff-target binding sites of site-directed polypeptides.

Sequence determination will be performed using methods that determinemany (typically thousands to billions) nucleic acid sequences in anintrinsically parallel manner, where many sequences are read outpreferably in parallel using a high throughput serial process. Suchmethods can include but are not limited to pyrosequencing (for example,as commercialized by 454 Life Sciences, Inc., Branford, Conn.);sequencing by ligation (for example, as commercialized in the SOLiD™technology, Life Technology, Inc., Carlsbad, Calif.); sequencing bysynthesis using modified nucleotides (such as commercialized in TruSeq™and HiSeg™ technology by Illumina, Inc., San Diego, Calif., HeliScope™by Helicos Biosciences Corporation, Cambridge, Mass., and PacBio RS byPacific Biosciences of California, Inc., Menlo Park, Calif.), sequencingby ion detection technologies (Ion Torrent, Inc., South San Francisco,Calif.); sequencing of DNA nanoballs (Complete Genomics, Inc., MountainView, Calif.); nanopore-based sequencing technologies (for example, asdeveloped by Oxford Nanopore Technologies, LTD, Oxford, UK), capillarysequencing (e.g, such as commercialized in MegaBACE by MolecularDynamics), electronic sequencing, single molecule sequencing (e.g., suchas commercialized in SMRT™ technology by Pacific Biosciences, MenloPark, Calif.), droplet microfluidic sequencing, sequencing byhybridization (such as commercialized by Affymetrix, Santa Clara,Calif.), bisulfate sequencing, and other known highly parallelizedsequencing methods.

In some embodiments, sequencing will be performed by microarrayanalysis.

Example 8 Generation of Antibody Libraries

The methods disclosed herein will be used to generate protein libraries(e.g., antibody libraries). Protein libraries will be useful forpreparing expression libraries, which will be used for screeningproteins (e.g. antibodies) for use in therapeutics, reagents, and/ordiagnostics. Protein libraries will also be useful for synthesizingand/or cloning additional antibodies.

Protein libraries will be generated by engineering a nucleicacid-targeting nucleic acid to hybridize to target nucleic acidsequences encoding immunoglobulins. The complexes comprising asite-directed polypeptide and the nucleic acid-targeting nucleic acidwill be purified using methods described herein. In some embodiments,the nucleic acid hybridizing to the nucleic acid-targeting nucleic acidwill be the target nucleic acid and will be eluted and sequenced, usingmethods described herein. In some embodiments, the nucleic acidhybridizing to the nucleic-acid targeting nucleic acid will not be thetarget nucleic acid. The target nucleic acid will be the nucleic acidthat is excised between the cleavage sites of a plurality of complexes(e.g., complexes comprising a site-directed polypeptide and nucleicacid-targeting nucleic acid). The excised target nucleic acid will bepurified and sequenced, using methods described herein.

Example 9 Genotyping

The methods disclosed herein will be used to perform Human LeukocyteAntigen (HLA) typing. HLA genes are some of the most polymorphic genesin humans. Understanding the genotypes of these regions will beimportant for obtaining a good match for tissue and organ transplants.

To perform HLA typing, a nucleic acid-targeting nucleic acid will beengineered to hybridize to target nucleic acid sequences in HLA genes.The complexes comprising a site-directed polypeptide and the nucleicacid-targeting nucleic acid will be purified using methods describedherein. In some embodiments, the nucleic acid hybridizing to the nucleicacid-targeting nucleic acid will be the target nucleic acid and will beeluted and sequenced, using methods described herein. In someembodiments, the nucleic acid hybridizing to the nucleic-acid targetingnucleic acid will not be the target nucleic acid. The target nucleicacid will be the nucleic acid that is excised between the cleavage sitesof a plurality of complexes (e.g., complexes comprising a site-directedpolypeptide and nucleic acid-targeting nucleic acid). The excised targetnucleic acid will be purified and sequenced, using methods describedherein.

Example 10 Site-Directed Polypeptide Immunoprecipitation

The disclosure provides methods for nuclease immunoprecipitation andsequencing (NIP-Seq). In some embodiments, the method will comprise a)contacting a nucleic acid sample with an enzymatically inactivesite-directed polypeptide, wherein the enzymatically inactivesite-directed polypeptide binds a target nucleic acid, thereby forming acomplex, b) capturing the complex with a capture agent, and c)sequencing the target nucleic acid. In some embodiments, the method willfurther comprise d) determining the identity of the off-target bindingsite.

In some embodiments, the methods of the disclosure will be performedoutside of cells. For example, a sample will comprise purified genomicDNA.

The site-directed polypeptide-target nucleic acid complexes will befixed or cross-linked to form complexes.

The nucleic acid (e.g., genomic DNA) will be treated to fragment the DNAbefore affinity purification. Fragmentation can be performed throughphysical, mechanical or enzymatic methods. Physical fragmentation caninclude exposing a target polynucleotide to heat or to ultraviolet (UV)light. Mechanical disruption may be used to mechanically shear a targetpolynucleotide into fragments of the desired range. Mechanical shearingmay be accomplished through a number of methods known in the art,including repetitive pipetting of the target polynucleotide, sonicationand nebulization. Target polynucleotides may also be fragmented usingenzymatic methods. In some cases, enzymatic digestion may be performedusing enzymes such as using restriction enzymes. Restriction enzymes maybe used to perform specific or non-specific fragmentation of targetpolynucleotides. The methods may use one or more types of restrictionenzymes, generally described as Type I enzymes, Type II enzymes, and/orType III enzymes. Type II and Type III enzymes are generallycommercially available and well known in the art. Type II and Type IIIenzymes recognize specific sequences of nucleotide nucleotides within adouble stranded polynucleotide sequence (a “recognition sequence” or“recognition site”). Upon binding and recognition of these sequences,Type II and Type III enzymes cleave the polynucleotide sequence. In somecases, cleavage will result in a polynucleotide fragment with a portionof overhanging single stranded DNA, called a “sticky end.” In othercases, cleavage will not result in a fragment with an overhang, creatinga “blunt end.” The methods may comprise use of restriction enzymes thatgenerate either sticky ends or blunt ends.

Once fragmented, the complexes comprising the site-directed polypeptidewill be purified by incubation with a solid support For example, if thesite-directed polypeptide comprises a biotin tag, the solid support willbe coated with avidin or streptavidin to bind to the biotin tag.

In an alternative embodiment, once fragmented, the complexes comprisingthe site-directed polypeptide, the target nucleic acid, and/or thenucleic acid-targeting nucleic acid, will be purified by incubation witha capture agent. The capture agent will bind to the affinity tag fusedto the site-directed polypeptide. The capture agent will comprise anantibody. For example, if the affinity tag fused to the site-directedpolypeptide is a FLAG tag, then the capture agent will be ananti-FLAG-tag antibody.

The capture agent will be purified with a solid support. For example, ifthe capture agent comprises a biotin tag, the bead will be coated withavidin or streptavidin to bind the biotinylated capture agent.

In some embodiments of the method, two or more rounds of purificationwill be performed. A first round will comprise purification with a solidsupport that can bind to the affinity tag of the capture agent and asecond round will comprise purification with a solid support that canbind to the affinity tag of the site-directed polypeptide. A first roundwill comprise purification with a solid support that will bind to theaffinity tag of the site-directed polypeptide and a second round willcomprise purification with a solid support that will bind to theaffinity tag of the capture agent.

In some embodiments, the method will be used to optimize the bindingspecificity of a site-directed polypeptide by performing the method morethan once.

The captured complex will comprise site-directed polypeptide and atarget nucleic acid. The target nucleic acid will be eluted from thesite-directed polypeptide complex by standard methods including highsalt washing, ethanol precipitation, boiling, gel purification, and thelike.

The eluted DNA will be prepared for sequencing analysis using standardmethods. The sequencing libraries will be sequenced and analyzed toidentify the sequence, and frequency of nuclease-binding sites.

In some embodiments, the method will be performed a plurality of times.In some embodiments, the method further comprises collecting data andstoring data. The data can be stored collected and stored on a computerserver.

Example 11 In Vivo Site-Directed Polypeptide Immunoprecipitation

In some embodiments, the method will comprise: a) introducing anenzymatically inactive site-directed polypeptide into a cell, whereinthe enzymatically inactive site-directed polypeptide binds a targetnucleic acid, thereby forming a complex, b) capturing the complex with acapture agent, and c) sequencing the target nucleic acid. In someembodiments, the method will further comprise d) determining theidentity of the off-target binding site.

In some instances, the site-directed polypeptide will comprise anaffinity tag. Polypeptides comprising an affinity tag have beendescribed herein.

Cells will be fixed or cross-linked. Fixed and/or cross-linked cellswill be lysed. Lysis conditions will be chosen to maintain intactprotein-DNA complexes. The lysate will be treated to fragment the DNAbefore affinity purification. Suitable fragmentation techniques aredescribed herein.

Once fragmented, the complexes comprising the site-directed polypeptide,the target nucleic acid and/or the nucleic acid-targeting nucleic acid,will be purified from the lysate by incubation with a solid support. Forexample, if the site-directed polypeptide comprises a biotin tag, thesolid support will be coated with avidin or streptavidin to bind to thebiotin tag.

In an alternative embodiment, once fragmented, the complexes comprisingthe site-directed polypeptide, will be purified from the lysate byincubation with a capture agent. The capture agent will bind to theaffinity tag fused to the site-directed polypeptide. The capture agentwill comprise an antibody. For example, if the affinity tag fused to thesite-directed polypeptide is a FLAG tag, then the capture agent will bea FLAG-tag antibody.

In some embodiments, the capture agent will comprise an affinity tag.The capture agent will be purified with a solid support. The solidsupport will bind to the affinity tag of the capture agent. For example,if the capture agent comprises a biotin tag, the bead will be coatedwith avidin or streptavidin to bind the biotinylated capture agent.

In some embodiments of the method, two rounds of purification will beperformed. In some instances, a first round will comprise purificationwith a solid support that will bind to the affinity tag of the captureagent and a second round will comprise purification with a solid supportthat will bind to the affinity tag of the site-directed polypeptide. Insome instances, a first round will comprise purification with a solidsupport that will bind to the affinity tag of the site-directedpolypeptide, and a second round will comprise purification with a solidsupport that will bind to the affinity tag of the capture agent.

In some embodiments, the method will be used to optimize the bindingspecificity of a site-directed polypeptide by performing the method morethan once.

The captured complex will comprise site-directed polypeptide and atarget nucleic acid. The target nucleic acid will be eluted from thesite-directed polypeptide complex by standard methods including highsalt washing, ethanol precipitation, boiling, gel purification, and thelike.

The eluted DNA will be prepared for sequencing analysis. Sequencinglibraries will be made from the eluted target nucleic acid. Thesequencing libraries will be sequenced and analyzed to identify thesequence, and frequency of nuclease-binding sites.

Example 12 Immunoprecipitation with an Enzymatically InactiveEndoribonuclease Capture Agent

A method for determining the identity of an off-target binding site of anuclease will comprise: a) contacting a nucleic acid sample with aenzymatically inactive site-directed polypeptide and a nucleicacid-targeting nucleic acid, wherein the enzymatically inactivesite-directed polypeptide and nucleic acid-targeting nucleic acid bindsa target nucleic acid, thereby forming a complex, b) capturing thecomplex with a capture agent, wherein the capture agent comprises aconditionally enzymatically inactive site-directed polypeptide, c)sequencing the target nucleic acid, and d) determining the identity ofthe off-target binding site. This method is designed to be performed ona cell-free nucleic acid sample, and/or a nucleic acid sampleoriginating from a cell.

The site-directed polypeptide-target nucleic acid complexes will befixed or cross-linked to form complexes.

If the nucleic acid sample originates from cells, the fixed and/orcross-linked complexes will be lysed. Lysis conditions can be chosen tomaintain intact protein-DNA complexes. The lysate will be treated tofragment the DNA before affinity purification. If the nucleic acidsample originates from a cell-free sample, the cell-free nucleic acidwill be treated to fragment the DNA. Suitable fragmentation techniquesare described herein.

In some embodiments, the nucleic acid-targeting nucleic acid willcomprise an affinity tag. The affinity tag will comprise a sequence thatcan bind to a conditionally enzymatically inactive site-directedpolypeptide. In some instances, the affinity tag will comprise asequence that can bind to a conditionally enzymatically inactiveendoribonuclease. In some instances, the affinity tag will comprise asequence that can bind to a conditionally enzymatically inactive Csy4protein. The conditionally enzymatically inactive site-directedpolypeptide will bind, but not cleave, the affinity tag. The affinitytag will comprise the nucleotide sequence5′-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3′. The affinity tag will be introducedinto a nucleic acid using standard recombinant methods.

Once fragmented, the complexes comprising the site-directed polypeptide,the target nucleic acid, and/or the nucleic acid-targeting nucleic acidwill be purified by incubation with a conditionally enzymaticallyinactive site-directed polypeptide (e.g., variant Csy4).

In some embodiments, the conditionally enzymatically inactivesite-directed polypeptide will comprise an affinity tag.

The conditionally enzymatically inactive site-directed polypeptide willbe purified with a solid support. The solid support will bind to theaffinity tag of the conditionally enzymatically inactive site-directedpolypeptide. For example, if the conditionally enzymatically inactivesite-directed polypeptide comprises a biotin tag, the bead will becoated with avidin or streptavidin to bind the biotinylated captureagent.

In some embodiments, the enzymatically inactive site-directedpolypeptide will be immobilized on any of a variety of insolublesupport.

In some embodiments of the method, two rounds of purification will beperformed. In some instances, a first round will comprise purificationwith a solid support that will bind to the affinity tag of theconditionally enzymatically inactive site-directed polypeptide (e.g.,variant Csy4) and a second round will comprise purification with a solidsupport that will bind to the affinity tag of the site-directedpolypeptide. In some instances, a first round will comprise purificationwith a solid support that will bind to the affinity tag of thesite-directed polypeptide and a second round will comprise purificationwith a solid support that will bind to the affinity tag of theconditionally enzymatically inactive site-directed polypeptide (e.g.,variant Csy4).

In some embodiments, the method will be used to optimize the bindingspecificity of a site-directed polypeptide by performing the method morethan once.

The captured complex will comprise a site-directed polypeptide and atarget nucleic acid. The target nucleic acid will be eluted from thesite-directed polypeptide complex by standard methods including highsalt washing, ethanol precipitation, boiling, gel purification, and thelike.

The eluted DNA will be prepared for sequencing analysis. The sequencinglibraries will be sequenced and analyzed to identify the sequence, andfrequency of nuclease-binding sites.

Example 13 Sequencing

The eluted target nucleic acids will be prepared for sequencinganalysis. Preparation for sequencing analysis will include thegeneration of sequencing libraries of the eluted target nucleic acid.Sequencing analysis will determine the identity and frequency ofoff-target binding sites of site-directed polypeptides.

Sequence determination will also be performed using methods thatdetermine many (typically thousands to billions) nucleic acid sequencesin an intrinsically parallel manner, where many sequences are read outpreferably in parallel using a high throughput serial process. Suchmethods include but are not limited to pyrosequencing (for example, ascommercialized by 454 Life Sciences, Inc., Branford, Conn.); sequencingby ligation (for example, as commercialized in the SOLiD™ technology,Life Technology, Inc., Carlsbad, Calif.); sequencing by synthesis usingmodified nucleotides (such as commercialized in TruSeq™ and HiSeg™technology by Illumina, Inc., San Diego, Calif., HeliScope™ by HelicosBiosciences Corporation, Cambridge, Mass., and PacBio RS by PacificBiosciences of California, Inc., Menlo Park, Calif.), sequencing by iondetection technologies (Ion Torrent, Inc., South San Francisco, Calif.);sequencing of DNA nanoballs (Complete Genomics, Inc., Mountain View,Calif.); nanopore-based sequencing technologies (for example, asdeveloped by Oxford Nanopore Technologies, LTD, Oxford, UK), and otherknown highly parallelized sequencing methods.

Example 14 Modification of a Target Nucleic Acid with an EffectorProtein

A vector comprising a site-directed polypeptide, a nucleicacid-targeting nucleic acid, and/or an effector protein is introducedinto a cell. Once inside the cell a complex is formed comprising theelements encoded in the vector. The nucleic acid-targeting nucleic acidis modified with a Csy4 protein binding sequence. The effector protein,Csy4, binds to the modified nucleic acid-targeting nucleic acid. Csy4comprises a non-native sequence (e.g., a fusion), that modifies a targetnucleic acid. The non-native sequence is a sequence that modifies thetranscription of the target nucleic acid. The non-native sequence is atranscription factor. The transcription factor increases the level oftranscription of the target nucleic acid. In some cases, the non-nativesequence is a methylase. The methylase results in increases inmethylation of the target nucleic acid. In some cases the non-nativesequence is a demethylase. The demethylase results in decreases inmethylation of the target nucleic acid. In some cases, the non-nativesequence is a Rad51-recruiting peptide. The Rad51-recruiting peptideincreases the level of homologous recombination at the target site. Insome cases, the non-native sequence is a BCRA-2 recruiting peptide. TheBRCA-2-recruiting peptide increases the level of homologousrecombination at the target site.

Example 15 Use of a Site-Directed Polypeptide as a Biosensor for aGenetic Mobility Event

A vector(s) comprising a site-directed polypeptide, a nucleicacid-targeting nucleic acid, and/or an effector protein is introducedinto a cell. The site-directed polypeptide and effector proteins arefused to cellular localization sequences (e.g. a nuclear localizationsignal). Once inside the cell a complex is formed comprising theelements encoded in the vector(s). In some instances, two vectors areintroduced into the cell. The vector(s) encodes for a first effectorprotein (Csy4) that comprises a first inactive portion of a split greenfluorescent protein (GFP) and binds to a first nucleic acid-targetingnucleic acid and a second effector protein (Csy4, Cas5, or Cas6) thatcomprises a second inactive portion of the split GFP and binds to asecond nucleic acid-targeting nucleic acid. The first nucleicacid-targeting nucleic acid is modified with a first Csy4, Cas5 or Cas6protein binding sequence that can be bound by a first Csy4, Cas5 or Cas6protein. The second nucleic acid-targeting nucleic acid is modified witha second Csy4, Cas5 or Cas6 protein binding sequence that can be boundby a second Csy4, Cas5 or Cas6 protein. In some embodiments, the firstCsy4, Cas5 or Cas6 protein interacts with the first Csy4, Cas5 or Cas6protein binding sequence, and the second Csy4, Cas5 or Cas6 proteininteracts with the second Csy4, Cas5 or Cas6 protein binding sequence.When the first and second nucleic acid-targeting nucleic acids directthe site-directed polypeptide to bind to two sequences that are in closeproximity, the first effector protein and the second effector proteinwill bring the first inactive portion of the split GFP into contact withthe second inactive portion of the split GFP, to generate an active GFP.The nucleic acid-targeting nucleic acids of the complex are designedsuch that one nucleic acid-targeting nucleic acid guides the complex to,for example, a region at or near the Bcr gene, and another nucleicacid-targeting nucleic acid guides the complex to, for example, a regionat or near the Abl gene. If a translocation event has not occurred theBcr gene is on chromosome 22 and the Abl gene is on chromosome 9, andthe target nucleic acid sequences are sufficiently far enough apart suchthat the two inactive portions of the split GFP system are unable tointeract, thereby not generating a signal. If a translocation event hasoccurred, the Bcr gene and the Abl gene are translocated such that thegenes are close together. In this instance, the target nucleic acidsequences are sufficiently close enough together such that the twoinactive portions of the split GFP system come together to form anactive GFP. A GFP signal can be detected by a fluorometer. The signal isindicative of a particular genotype resulting from the genetic mobilityevent.

Example 16 Use of a Site-Directed Polypeptide as a Biosensor for aGenetic Mutation

The system described in Example 2 can also be used to detect thepresence of specific mutation within a cell. In this example, a firstnucleic-acid targeting nucleic acid is chosen to direct the sitedirected polynucleotide to a native sequence located near a mutationsite. The second nucleic-acid targeting nucleic acid is chosen torecognize a mutant sequence (e.g., the mutant sequence having beenidentified by DNA sequencing). The nucleic-acid targeting nucleic acidis chosen such that the mutant sequence occurs within the first 12nucleic acids immediately 5′ to the PAM sequence in the site. In thisinstance, the target nucleic acid sequences are sufficiently closeenough together such that the two inactive portions of the split GFPsystem come together to form an active GFP. A GFP signal can be detectedby a fluorometer. The signal is indicative of a particular genotype

Example 17 Use of a Site-Directed Polypeptide as as a Therapeutic forDiseases that Comprise a Genetic Mobility Event

A vector (s) comprising a site-directed polypeptide, a nucleicacid-targeting nucleic acid, and/or an effector protein, a nucleic acidcomprising a cell-lysis inducing peptide (e.g. Adenovirus death protein)operably linked to a first promoter will also be introduced into thecell. Once inside the cell a complex is formed comprising the elementsencoded in the vector(s). In some instances, two vectors are introducedinto the cell. The vector (s) encodes for a first effector protein(comprising a first Csy4, Cas5 or Cas6 protein sequence) that binds to afirst nucleic acid-targeting nucleic acid and comprises an activatordomain for a first transcription factor that binds to the first promoterand a second effector protein (comprising a second Csy4, Cas5 or Cas6protein sequence) that binds to a second nucleic acid-targeting nucleicacid and comprises the DNA binding domain for the first transcriptionfactor. The first nucleic acid-targeting nucleic acid is modified with afirst Csy4, Cas5 or Cas6 protein binding sequence that can be bound by afirst Csy4, Cas5 or Cas6 protein sequence. The second nucleicacid-targeting nucleic acid is modified with a second Csy4, Cas5 or Cas6protein binding sequence that can be bound by a second Csy4, Cas5 orCas6 protein. In some embodiments, the first Csy4, Cas5 or Cas6 proteininteracts preferentially with the first Csy4, Cas5 or Cas6 proteinbinding sequence, and the second Csy4, Cas5 or Cas6 protein interactspreferentially with the second Csy4, Cas5 or Cas6 protein bindingsequence. If a diseased cell comprises a genome containing a geneticmobility event, when the first and second nucleic acid-targeting nucleicacids direct the site-directed polypeptide to bind to two sequences thatare in close proximity, the first effector protein and the secondeffector protein will bring the activator domain and the DNA-bindingdomain of the first transcription factor into close proximity. TheDNA-binding domain of the first transcription factor can bind to thefirst promoter operably linked to the cell-lysis inducing peptide, andthe proximal activator domain will induce transcription of RNA encodingthe cell-lysis inducing peptide. In a non-diseased cell, that does notcomprise the genetic mobility event, the DNA-binding domain and theactivator domains of the first transcription factor will not be broughtinto close proximity, and there will be no transcription of thecell-lysis inducing peptide.

The nucleic acid-targeting nucleic acids of the complex are designedsuch that one nucleic acid-targeting nucleic acid guides the complex to,for example, a region at or near the Bcr gene, and another nucleicacid-targeting nucleic acid guides the complex to, for example, a regionat or near the Abl gene. In a non-diseased cell, a translocation eventhas not occurred, the Bcr gene is on chromosome 22 and the Abl gene ison chromosome 9, and the target nucleic acid sequences are sufficientlyfar enough apart such that the two inactive portions of thetranscription factor system are unable to interact, and cannot inducetranscription of the cell-lysis inducing peptide. In a diseased cell, inwhich a translocation event has occurred, the Bcr gene and the Abl geneare translocated such that the genes are close together. In thisinstance, the target nucleic acid sequences are sufficiently closeenough together such that the two inactive portions of the transcriptionfactor system come together to induce transcription of the cell-deathinducing peptide. Cell-lysis will be dependent upon a particulargenotype resulting from the genetic mobility event.

Example 18 Use of a Site-Directed Polypeptide as a Therapeutic forDiseases that Comprise a Genetic Mutation

The system described in Example 4 can also be used to detect thepresence of specific mutation within a cell. In this example, the firstnucleic-acid targeting nucleic acid is chosen to direct the sitedirected polynucleotide to a native sequence located near a mutationsite. The second nucleic-acid targeting nucleic acid is chosen torecognize a mutant sequence (e.g., the mutant sequence having beenidentified by DNA sequencing). The nucleic-acid targeting nucleic acidis chosen such that the mutant sequence occurs within the first 12nucleic acids immediately 5′ to the PAM sequence in the site. In thisinstance, the target nucleic acid sequences are sufficiently closeenough together such that the two inactive portions of the transcriptionfactor come together to enable transcription of a cell-lysis inducingpeptide.

Example 19 Recruiting the Immune System to Attack Diseased TissueContaining a Genetic Mobility Event or a Genetic Mutation

The system described in Example 4 and/or 5 can also be used to directtranscription by the split transcription factor system that will resultin the display of an antigen on the cell surface. In some instances, theantigen is a peptide displayed by an MHC class II molecules. In someinstances, the antigen is a cell-surface protein that recruits immuneeffector cells to the site.

Example 20 Detecting Three-Dimensional Position of Nucleic Acids

A vector (s) comprising a site-directed polypeptide, a nucleicacid-targeting nucleic acid, and/or an effector protein is introducedinto a cell. Once inside the cell a complex is formed comprising theelements encoded in the vector(s). Two vectors are introduced into thecell. One vector encodes for an effector protein (Csy4) that comprises afirst inactive portion of a split affinity tag system. A second vectorencodes for an effector protein (Csy4, Cas5, or Cas6) that comprises asecond inactive portion of the split affinity tag. The nucleicacid-targeting nucleic acid of the complexes is modified with a Csy4,Cas5 or Cas6 protein binding sequence. The effector proteins bind to themodified nucleic acid-targeting nucleic acid. The nucleic acid-targetingnucleic acids are designed to guide the complexes to regions of interestin a three-dimensional nucleic acid structure (e.g., chromatin). If thetarget sequences are not close together in space, the two inactiveportions of the split affinity tag are unable to interact. If the targetsequences are close together in space, then the two inactive portions ofthe split affinity tag can come together to form the whole affinity tag.

The cells are lysed and the cell lysis is incubated with an antibodythat binds to the affinity tag. The antibody is purified, therebypurifying the affinity tag and the nucleic acid to which the complexesare bound. The purified nucleic acid is dissociated from the complexesusing high salt wash. The dissociated purified nucleic acid is preparedfor sequencing analysis, and sequenced. The sequencing resultscorrespond to regions of chromatin that are close together inthree-dimensional space. The sequencing results can be used to furtherunderstand gene expression and treat disease.

Example 21 Multiplex Genome Engineering

A vector comprising a multiplexed genetic targeting agent comprisingnucleic acid modules which comprise a nucleic acid-targeting nucleicacid and an endoribonuclease binding sequence is introduced into a cell.In some embodiments, the cell already comprises a site-directedpolypeptide and an endoribonuclease. In some instances, the cell iscontacted with a vector comprising a polynucleotide sequence encoding asite-directed polypeptide and a vector comprising a polynucleotidesequence encoding an endoribonuclease. In some instances, the cell iscontacted with a vector comprising a polynucleotide sequence encodingboth the site-directed polypeptide and the endoribonuclease. In someembodiments, the vector comprises a polynucleotide sequence encoding oneor more endoribonucleases. In some embodiments, the vector comprises apolynucleotide sequence encoding a multiplexed genetic targeting agent,a site-directed polypeptide, and one or more endoribonucleases. Thearray is transcribed into RNA. The one or more endoribonucleases bindsto the one or more endoribonuclease binding sequences in the multiplexedgenetic targeting agent. The one or more endoribonucleases cleaves theone or more endoribonuclease binding sequences in the multiplexedgenetic targeting agent, thus liberating the individual nucleic acidmodules. In some embodiments, the nucleic acid modules comprise all,some, or none, of the endoribonuclease binding sequence.

The liberated nucleic acid modules bind to site-directed polypeptides,thereby forming complexes. The complexes are targeted to one or moretarget nucleic acids. The one or more nucleic acid modules hybridizes tothe one or more target nucleic acids. The one or more site-directedpolypeptides cleaves the one or more target nucleic acids at a cleavagesite defined by the nucleic acid module, thus resulting in one or moremodified target nucleic acids.

In some embodiments, one or more donor polynucleotides and/or a vectorsencoding the same are introduced into the cell. One or more donorpolynucleotides are incorporated into the one or more cleaved targetnucleic acids, thereby resulting in one or more modified target nucleicacids (e.g., addition). In some instances, the same donor polynucleotideis incorporated into multiple cleavage sites. In some instances, one ormore donor polynucleotides are incorporated into multiple cleavagesites. In some instances, no donor polynucleotide and/or vector encodingthe same are introduced into the cells. In these instances, the modifiedtarget nucleic acid can comprise a deletion.

Example 22 Method of Stoichiometric Delivery of RNA to a Cell

In some embodiments, the disclosure provides for a method forstoichiometric delivery of nucleic acids to the nucleus of a cell. Insome embodiments, three stoichiometrically deliverable nucleic acid areused: one encoding for Cas9, one encoding for a nucleic-acid targetingnucleic acid, and one encoding Csy4. Each of the three nucleic acidscomprises a Csy4-binding site.

In some embodiments, the method provides for a tandem fusionpolypeptide. The fusion polypeptide comprises three Csy4 polypeptides.The three Csy4 polypeptides are separated by a linker. The three Csy4polypeptides bind to the Csy4-binding sites on each of the three nucleicacid molecules, thereby forming a complex.

In some embodiments, the complex is formed outside of a cell andintroduced into the cell. The complex is formed by mixing the threestoichiometrically deliverable nucleic acids and the fusion protein andletting the reaction occur to allow binding between the tandem fusionpolypeptide and three Csy4-binding sites. The complex is introduced byinjection, electroporation, transfection, transformation, viraltransduction, and the like. Inside the cell, some of the nucleic acidsof the complex are translated. In some embodiments, the resultingtranslation products are Csy4 and NLS-Cas9 (e.g., Cas9 comprising anNLS. The NLS may not have to be at the N-terminus). The Csy4 cleaves theCsy4-binding site on the nucleic acid encoding the nucleicacid-targeting nucleic acid, thereby liberating the nucleicacid-targeting nucleic acid from the tandem fusion polypeptide. NLS-Cas9binds the liberated nucleic acid-targeting nucleic acid, thereby forminga unit. This unit translocates to the nucleus. Inside the nucleus, theunit is guided to a target nucleic acid that hybridizes with the spacerof the nucleic acid-targeting nucleic acid. The Cas9 of the unit cleavesthe target nucleic acid. The cleavage of the target nucleic acid by Cas9is referred to as genome engineering.

In some embodiments, the complex is formed inside of a cell. A vectorencoding the three stoichiometrically deliverable nucleic acids isintroduced into the cell. Three different vectors encoding one of eachof the three stoichiometrically deliverable nucleic acids is introducedthe cell. Two vectors are introduced into the cell, wherein one of thetwo vectors encodes for two stoichiometrically deliverable nucleic acidsand one of the two vectors encodes for one stoichiometricallydeliverable nucleic acid. Any of the vectors can encode the tandemfusion polypeptide.

Inside the cell, vector nucleic acid encoding RNA or a polypeptide istranscribed into RNA. A complex comprising the three nucleic acids andthe tandem fusion polypeptide is formed, whereby each of theCsy4-binding proteins binds to the Csy4-binding site on each of thethree nucleic acids. The nucleic acids of the complex are translated. Insome embodiments, the resulting translation products are Csy4 andNLS-Cas9 (e.g., Cas9 comprising an NLS. The NLS may not have to be atthe N-terminus). The Csy4 cleaves the Csy4-binding site on the nucleicacid encoding the nucleic acid-targeting nucleic acid, therebyliberating the nucleic acid-targeting nucleic acid from the tandemfusion polypeptide. NLS-Cas9 binds the liberated nucleic acid-targetingnucleic acid, thereby forming a unit. This unit translocates to thenucleus. Inside the nucleus, the unit is guided to a target nucleic acidthat hybridizes with the spacer of the nucleic acid-targeting nucleicacid. The Cas9 of the unit cleaves the target nucleic acid.

Example 23 Method of Stoichiometric Delivery of Multiple NucleicAcid-Targeting Nucleic Acids to a Cell

In some embodiments, the disclosure provides for a method forstoichiometric delivery of a plurality of nucleic acids to a cell,wherein some of the plurality of nucleic acids are nucleicacid-targeting nucleic acids. In some embodiments, the plurality ofnucleic acids comprises four nucleic acids: one encoding for Cas9, twoencoding for nucleic-acid targeting nucleic acids, and one encoding forCsy4.

The nucleic acids comprise two or more nucleic acid-binding proteinbinding sites. In some instances, the first nucleic acid-binding proteinbinding site (e.g., the more 5′ site) comprises a Csy4 binding site. Insome instances, the second nucleic acid-binding protein binding site(e.g., the more 3′ site) comprises a different nucleic acid-bindingprotein binding site (e.g., MS2 binding site). In some instances, thesecond nucleic acid-binding protein binding site from each of thestoichiometrically deliverable nucleic acids is different. For example,the second nucleic acid-binding protein binding site can be a site thatbinds one of a CRISPR polypeptide (e.g., Cas5, Cas6) In some instances,the nucleic acid encoding Cas9 also comprises a nuclear localizationsignal (NLS).

In some embodiments, the tandem fusion polypeptide comprises threenucleic acid-binding proteins. The three nucleic acid-binding proteinsare Csy4, Cas5, Cas6. The tandem fusion polypeptide comprises a nuclearlocalization signal. The tandem fusion polypeptides comprises more thanone copy of a nucleic acid-binding protein (e.g., 2 copies of Csy4, onecopy of Cas5, one copy of Cas6).

In some embodiments, a complex comprising the tandem fusion protein andthe four nucleic acids is formed outside of a cell. The complex isformed by mixing the four nucleic acids and the tandem fusion proteinand letting the reaction occur to allow binding between the tandemfusion polypeptide and four nucleic acid-binding protein binding sites.The complex is introduced into the cell. Introduction occurs by eithertransformation, transfection, viral transduction, microinjection, orelectroporation, or any technique capable of introducing biomoleculesacross a cell membrane. The complex is formed inside the cell (e.g.,after introduction of vectors comprising nucleic acid sequences encodingthe nucleic acids and tandem fusion protein).

Inside the cell, the nucleic acids encoding Csy4 and Cas9 aretranslated, resulting in Csy4 and NLS-Cas9 (e.g., Cas9 comprising anNLS. The NLS may not have to be at the N-terminus). Csy4 cleaves theCsy4-binding site on the nucleic acids encoding the nucleicacid-targeting nucleic acids, thereby liberating them from the tandemfusion polypeptide.

NLS-Cas9 binds the liberated nucleic acid-targeting nucleic acids,thereby forming a plurality units. The units translocate to the nucleus.Inside the nucleus, the units are guided to a target nucleic acid thathybridizes with the spacer of the nucleic acid-targeting nucleic acid ofthe unit. The Cas9 of the unit cleaves the target nucleic acid.

Example 24 Method of Stoichiometric Delivery of RNA and a DonorPolynucleotide

The disclosure provides for a method of stoichiometric delivery of RNAcomponents that can be used in genome engineering. The method can alsocomprise delivery of a donor polynucleotide that can be inserted into asite of genome engineering.

The disclosure provides for a method for stoichiometric delivery of aplurality of RNAs and a donor polynucleotide to a cell. In someembodiments, the plurality of RNAs comprises three RNAs and a DNA. Insome instances, the three RNAs are: one encoding for Cas9, one encodingfor Csy4, and one encoding for a nucleic acid-targeting nucleic acid.The DNA is a DNA encoding a donor polynucleotide.

In some instances, the RNAs comprise a plurality of nucleic acid-bindingprotein binding sites (e.g., two nucleic acid-binding protein bindingsites). The first nucleic acid-binding protein binding site (e.g., themore 5′ site) comprises a Csy4 binding site. The second nucleicacid-binding protein binding site (e.g., the more 3′ site comprises adifferent nucleic acid-binding protein binding site). The second nucleicacid-binding protein binding site in each of the nucleic acids of thedisclosure is different. For example, the second nucleic acid-bindingprotein binding site binds a CRISPR polypeptide (e.g., Cas5, Cas6)and/or a DNA binding protein (e.g., a zinc finger protein). The nucleicacids of the method also comprise a sequence encoding for a nuclearlocalization signal (e.g., the RNA encoding Cas9, and the DNA encoding adonor polynucleotide).

In some embodiments, the tandem fusion polypeptide comprises fournucleic acid-binding proteins (e.g., RNA-binding proteins andDNA-binding proteins). In some instances, three nucleic acid-bindingproteins are Csy4, Cas5, Cas6, and the fourth nucleic acid-bindingprotein is a DNA-binding protein (e.g., zinc finger protein). In someinstances, the tandem fusion polypeptide comprises a nuclearlocalization signal.

In some embodiments, a complex comprising the tandem fusion protein andthe nucleic acids (e.g., three RNAs and one DNA) is formed outside of acell. The complex is formed by mixing the nucleic acids (e.g., threeRNAs and one DNA) and the tandem fusion protein and letting the reactionoccur to allow binding between the tandem fusion polypeptide and fourRNA-binding protein binding sites. The complex can be introduced intothe cell. The complex is formed inside the cell (e.g., afterintroduction of vectors comprising nucleic acid sequences encoding thenucleic acids and tandem fusion protein).

Inside the cell, the RNAs encoding Csy4 and Cas9 are translated,resulting in Csy4 and NLS-Cas9 (e.g., Cas9 comprising an NLS. The NLSmay not have to be at the N-terminus as written here). Csy4 can cleavethe Csy4-binding site on the RNAs encoding the nucleic acid-targetingnucleic acid and the DNA, thereby liberating them from the tandem fusionpolypeptide. In some instances, the liberated donor polynucleotidetranslocates to the nucleus by its nuclear localization signal.

NLS-Cas9 binds the liberated nucleic acid-targeting nucleic acid,thereby forming a unit. The unit translocates to the nucleus. Inside thenucleus, the unit is guided to a target nucleic acid that hybridizeswith the spacer of the nucleic acid-targeting nucleic acid of the unit.The Cas9 of the unit cleaves the target nucleic acid. The donorpolynucleotide, a portion of the donor polynucleotide, a copy of thedonor polynucleotide, or a portion of a copy of the donor polynucleotidecan be inserted into the cleaved target nucleic acid.

Example 25 Seamless Selection of Genetically Modified Cells

A plurality of cells is contacted with a vector comprising sequencesencoding a polypeptide homologous to Cas9, a nucleic acid-targetingnucleic acid and a donor polynucleotide. In some cases, one or more ofthe sequences encoding the polypeptide homologous to Cas9, the nucleicacid-targeting nucleic acid and the donor polynucleotide are located ondifferent vectors. e cells are transfected with the vector. In someinstances, the cells are infected with a virus carrying the vector. Insome instances, the cell already comprises a protein homologous to Cas9and the vector does not encode this polypeptide. In some instances, thecell already comprises a CRISPR system (e.g. Cas proteins, crRNA andtracrRNA) and the vector only encodes the donor polynucleotide. Thedonor polynucleotide comprises sequences encoding a genetic element ofinterest and a reporter element. The reporter element comprises nucleicacid-targeting nucleic acid sequences, a protein homologous to Cas 9 anda fluorescent protein. The nucleic acid-targeting nucleic acid guideCas9 to a target nucleic acid (e.g. a site in the host cell genome),resulting in a double stranded DNA break of the target nucleic acid andinsertion of the donor polynucleotide. Insertion of the donorpolynucleotide is screened for by screening for the reporter. In somecases, screening comprises fluorescence-activated cell sorting.Screening comprises multiple selection methods. Cas9 and/or the nucleicacid-targeting nucleic acids are controlled by an inducible promoter.After selecting a population of cells that comprise the reporter signal,the reporter element is removed by activating the inducible promoter,which transcribes the nucleic acid-targeting nucleic acids and thesite-directed polypeptide of the donor polynucleotide. The transcribednucleic acid-targeting nucleic acids and the transcribed site-directedpolypeptide can form complexes. One complex can be targeted to the 3′end of the reporter element of the donor polynucleotide. One complex canbe targeted to the 5′ end of the reporter element of the donorpolynucleotide. The 3′ and 5′ ends of the reporter element can becleaved. The cleaved target nucleic acid can be rejoined by cellularmechanisms, thereby resulting in an in-frame nucleic acid sequenceencoding the same nucleic acid sequence as prior to insertion of thedonor polynucleotide. In this way, the reporter element is seamlesslyinserted and removed from cells.

Example 26 Method for Cas9 Cleavage Using Engineered NucleicAcid-Targeting Nucleic Acids

Reagents

pCB002 plasmid containing temp3 target DNA sequence was digested with 1Uof AscI per 1 ug of DNA to linearize the vector. The reaction wasstopped by incubating reaction mix at 80 C for 20 min. Reaction was thenpurified using Qiagen PCR clean up kit.

Single guide nucleic acid were generated using T7 High Yield RNASynthesis Kit (Cat No. E2040S), using half the volume of reagentsrecommended by the manufactures for RNA of >300 nucleotides. Generallybetween 200-350 ng of template were used in a 20 uL reaction thatincubates for 16 hours. Samples were treated with DNase, and purifiedusing Thermo GeneJet RNA purification Kit (Cat No. K0732), and eluted in20 uL. Typical yields range from 1.4-2 ug/uL.

At the start of cleavage assay set-up, all sgRNA were diluted to 3500 nMconcentration and heat shocked at 80 C for 15 minutes. Samples wereremoved from heating element and allowed to equilibrate to roomtemperature. An aliquot of 4 uL of single guide nucleic acid-targetingnucleic acid can be run out on an agarose gel to confirm RNA integrity.

Aliquots of Cas9 at 2-2.5 mg/mL were removed from freezer and thawed asquickly as possible and then diluted in 1× cleavage buffer toappropriate stock concentration.

Cleavage Assay

A master mix of water, 5× cleavage buffer of (100 mM HEPES, 500 mM KCl,25 mM MgCl2, 5 mM DTT, and 25% glycerol at pH 7.4), and Cas9 to 250 nMwas aliquoted into thin wall PCR tubes. sgRNA was added to appropriatetubes at a final concentration of 250 nM.

The reaction was incubated at 37 C for 10 minutes, 10 nM linearizedplasmid was added and reactions (final reaction volume 20 uL) wereincubated at 37 C for and hour. The reaction was terminated by heatingreaction to 60 C for 20 minutes. 10 uL aliquots were mixed with 2 uL6×DNA loading dye and analyzed by electrophoresis on a 1.5% agarose gelstained with SYBR safe. The appearance of a ˜2800 bp and ˜1300 bpfragments was indicative of Cas9 mediated cleavage. The results of theexperiments are shown in FIGS. 6, 10 and 11. All synthetic guide RNAsequences designed and shown in FIG. 5 supported sgRNA cleavage, exceptfor SGRv8, in which the entire complementary region was inverted. Theseresults indicate that different regions of the sgRNA can be amenable toengineering and still retain function.

Engineered nucleic acid-targeting nucleic acids were tested in the assaydescribed above. FIGS. 22A and 22B shows the designs of an initial setof duplex variants in single-guide nucleic acid-targeting nucleic acidbackbone variants used to test targeting and cleavage activity.

FIGS. 23A and 23B illustrate a second set of duplex variants withsmaller modifications in the duplex. V28 comprises a 2 base insertion 3′to the complementary region; V29 comprises a 3 base deletion 3′ to thecomplementary region.

FIGS. 24A and 24B illustrate tracr variants which comprise mutations inthe tracrRNA portion of the nucleic acid-targeting nucleic acid (i.e.,minimum tracrRNA sequence and 3′ tracrRNA sequence). V38-V41 comprisefusions between the complementary region/duplex and the 3′ ends of thetracr nucleic acid sequences from M. mobile 163K (v38), S. thermophilusLMD-9 (V39), C. jejuni (V40), and N. meningitides.

FIGS. 25A and 25B depict variants comprising modifications to enableCsy4 binding to the nucleic acid-targeting nucleic acid. The additionalhairpin sequences are derived from the CRISPR repeat in Pseudomonasaeruginosa PA14.

FIG. 26A-C shows data from the in vitro cleavage assay demonstrating theactivity of the nucleic acid-targeting nucleic acid variants on Cas9cleavage. Variant SGRv8 failed to support target nucleic acid cleavage(FIG. 26B lane 9).

FIG. 27 and FIG. 28 shows two independent repeats of the Cas9 cleavageassay testing the variants depicted in FIGS. 23-25.

Additional engineered nucleic acid-targeting nucleic acids were made aslisted in Table 2, and tested in the same assay. The results of theassay are shown in FIG. 37 and listed in the activity column of Table 2.

The results of these experiments indicate the importance of the bulgeand P-domain regions in effecting target nucleic acid cleavage. Thefunctionality of variants 42-45 indicates that the addition of a Csy4binding sequence to a nucleic acid-targeting nucleic acid does notdisrupt target nucleic acid cleavage.

Example 27 Sequencing Analysis Systems

FIG. 30 depicts a system that is configured to implement the methods ofthe disclosure. The system can include a computer server (“server”) thatis programmed to implement the methods described herein. FIG. 30 depictsa system 3000 adapted to enable a user to detect, analyze, andcommunicate sequencing results of for example, nuclease-targetedenriched nucleic acids, sequenced target nucleic acids, data concerningthe methods of the disclosure, diagnose a disease, genotype a patient,make a patient-specific treatment decision, or any combination thereof.The system 3000 includes a central computer server 3001 that isprogrammed to implement exemplary methods described herein. The server3001 includes a central processing unit (CPU, also “processor”) 3005which can be a single core processor, a multi core processor, orplurality of processors for parallel processing. The server 3001 alsoincludes memory 3010 (e.g. random access memory, read-only memory, flashmemory); electronic storage unit 3015 (e.g. hard disk); communicationsinterface 3020 (e.g. network adaptor) for communicating with one or moreother systems; and peripheral devices 3025 which may include cache,other memory, data storage, and/or electronic display adaptors. Thememory 3010, storage unit 3015, interface 3020, and peripheral devices3025 are in communication with the processor 3005 through acommunications bus (solid lines), such as a motherboard. The storageunit 3015 can be a data storage unit for storing data. The server 3001is operatively coupled to a computer network (“network”) 3030 with theaid of the communications interface 3020. The network 3030 can be theInternet, an intranet and/or an extranet, an intranet and/or extranetthat is in communication with the Internet, a telecommunication or datanetwork. The network 3030 in some cases, with the aid of the server3001, can implement a peer-to-peer network, which may enable devicescoupled to the server 3001 to behave as a client or a server. Themicroscope and micromanipulator can be peripheral devices 3025 or remotecomputer systems 3040.

The storage unit 3015 can store files, such as sequencing results,target binding sites, personalized genetic data, genotypes, images, dataanalysis of images and/or sequencing results, or any aspect of dataassociated with the disclosure.

The server can communicate with one or more remote computer systemsthrough the network 3030. The one or more remote computer systems maybe, for example, personal computers, laptops, tablets, telephones, Smartphones, or personal digital assistants.

In some situations the system 3000 includes a single server 3001. Inother situations, the system includes multiple servers in communicationwith one another through an intranet, extranet and/or the Internet.

The server 3001 can be adapted to store sequencing results, targetbinding sites, personalized genetic data, and/or other information ofpotential relevance. Such information can be stored on the storage unit3015 or the server 3001 and such data can be transmitted through anetwork.

Methods as described herein can be implemented by way of machine (orcomputer processor) executable code (or software) stored on anelectronic storage location of the server 3001, such as, for example, onthe memory 3010, or electronic storage unit 3015. During use, the codecan be executed by the processor 3005. In some cases, the code can beretrieved from the storage unit 3015 and stored on the memory 3010 forready access by the processor 3005. In some situations, the electronicstorage unit 3015 can be precluded, and machine-executable instructionsare stored on memory 3010. Alternatively, the code can be executed on asecond computer system 3040.

Aspects of the systems and methods provided herein, such as the server3001, can be embodied in programming. Various aspects of the technologymay be thought of as “products” or “articles of manufacture” typicallyin the form of machine (or processor) executable code and/or associateddata that is carried on or embodied in a type of machine readablemedium. Machine-executable code can be stored on an electronic storageunit, such memory (e.g., read-only memory, random-access memory, flashmemory) or a hard disk. “Storage” type media can include any or all ofthe tangible memory of the computers, processors or the like, orassociated modules thereof, such as various semiconductor memories, tapedrives, disk drives and the like, which may provide non-transitorystorage at any time for the software programming. All or portions of thesoftware may at times be communicated through the Internet or variousother telecommunication networks. Such communications, for example, mayenable loading of the software from one computer or processor intoanother, for example, from a management server or host computer into thecomputer platform of an application server. Thus, another type of mediathat may bear the software elements includes optical, electrical, andelectromagnetic waves, such as used across physical interfaces betweenlocal devices, through wired and optical landline networks and overvarious air-links. The physical elements that carry such waves, such aswired or wireless likes, optical links, or the like, also may beconsidered as media bearing the software. As used herein, unlessrestricted to non-transitory, tangible “storage” media, terms such ascomputer or machine “readable medium” refer to any medium thatparticipates in providing instructions to a processor for execution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, tangible storage medium,a carrier wave medium, or physical transmission medium. Non-volatilestorage media can include, for example, optical or magnetic disks, suchas any of the storage devices in any computer(s) or the like, such maybe used to implement the system. Tangible transmission media caninclude: coaxial cables, copper wires, and fiber optics (including thewires that comprise a bus within a computer system). Carrier-wavetransmission media may take the form of electric or electromagneticsignals, or acoustic or light waves such as those generated during radiofrequency (RF) and infrared (IR) data communications. Common forms ofcomputer-readable media therefore include, for example: a floppy disk, aflexible disk, hard disk, magnetic tape, any other magnetic medium, aCD-ROM, DVD, DVD-ROM, any other optical medium, punch cards, paper tame,any other physical storage medium with patterns of holes, a RAM, a ROM,a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, acarrier wave transporting data or instructions, cables, or linkstransporting such carrier wave, or any other medium from which acomputer may read programming code and/or data. Many of these forms ofcomputer readable media may be involved in carrying one or moresequences of one or more instructions to a processor for execution.

Example 28 Array-Based Sequencing Using a Site-Directed Polypeptide

A nucleic acid sample is ligated with a nucleic acid tag comprising asingle guide RNA and a detectable label. Together, the nucleic acidsample ligated to the nuclei acid tag is referred to as a tagged testsample. The tagged test sample is contacted to a microarray comprisingimmobilized oligonucleotides. The immobilized oligonucleotides are adouble stranded nucleic acid library. The oligonucleotides comprise adetectable label (e.g., fluorescent label. The individual members of thetagged test sample hybridize to the oligonucleotides to which they shareenough complementarity to facilitate hybridization. The amount ofhybridization can be quantified by comparing the intensities of the twodetectable labels from the sample library and the immobilizedoligonucleotides. For example, hybridized oligonucleotides can displaytwo detectable labels (that from the sample library and theoligonucleotide). Unhybridized oligonucleotides can display onedetectable label (that from the oligonucleotide). The hybridized probesare contacted with Cas9. Cas9 cleaves the oligonucleotides in themicroarray that have hybridized with members of the tagged test sample.Cleavage by the site-directed polypeptide allows the hybridized membersof the tagged test sample to be removed. After cleavage by thesite-directed polypeptide, only unhybridized oligonucleotide detectablelabels remain on the microarray. The remaining detectable label isquantified. The quantification of the remaining detectable labels iscorrelated to which sequences were represented in the nucleic acidsample and which were not (e.g., by position mapping). Oligonucleotidesthat do not display a remaining detectable label correspond to sequencesthat were represented in the nucleic acid sample. Oligonucleotides thatdisplay a remaining detectable label correspond to sequences that werenot represented in the nucleic acid sample.

Example 29 Cleavage of a Target Nucleic Acid with a Tagged NucleicAcid-Targeting Nucleic Acid

This example describes the results of target nucleic acid cleavage witha nucleic acid-targeting nucleic acid comprising a Csy4 binding sequenceon the 5′ end of the nucleic acid-targeting nucleic acid. Cas9 wasincubated with or without a guide RNA targeting a single site in alinear double stranded DNA sequence. After 1 hour, cleavage productswere separated and visualized on an agarose gel. FIG. 13D illustratesthat Cas9 cleavage mediated by a tagged nucleic acid-targeting nucleicacid (lane 3) was less efficient than Cas9 cleavage mediated by anuntagged nucleic acid-targeting nucleic acid (lane 1). After 1 hour,˜100% of the target was cleaved by Cas9 guided by the untagged nucleicacid-targeting nucleic acid, where as only a small fraction of thetarget was cleaved in the same time by Cas9 guided by the tagged nucleicacid-targeting nucleic acid. These experiments indicate the location ofthe non-native sequence can be used to tune cleavage effectiveness ofcleavage of the Cas9: nucleic acid-targeting nucleic acid complex. Forexample, FIG. 27 and FIG. 29 show the functionality of the addition of aCsy4 binding sequence to various locations in the nucleic acid-targetingnucleic acid that retain activity.

Example 30 Genome Engineering Genes in Blood Disorders

A nucleic acid-targeting nucleic acid comprising a spacer sequencedescribed in Figure XX is introduced into a cell with a site-directedpolypeptide, thereby forming a complex. The complex targets the geneinvolved in a blood disorder that has substantial complementarity to thespacer sequence of the nucleic acid-targeting nucleic acid. Once thenucleic acid-targeting nucleic acid is hybridized to the target nucleicacid the site-directed polypeptide cleaves the target nucleic acid. Thecleaved target nucleic acid is can be engineered with a donorpolynucleotide.

Example 31 Protocol to Determine Target Nucleic Acid Cleavage andModification

This protocol can be used to determine if a target nucleic acid has beencleaved or if the target nucleic acid has been modified such as with aninsertion or deletion. Primers surrounding the target site are used toPCR amplify, in a 25 μL reaction, a 500-600 nt product from gDNA. Theprimers comprise at least 100 nt on either side of the cut site. Theresulting products from the cleavage assay are about greater than 100nt.

About 5 μL of PCR product is run on an agarose gel to determine ifamplification is clean. With the remaining PCR product the melt andannealing protocol is as follows:

95° C. 10 min  95° C. to 85° C. (−2.0° C./s) 85° C. 1 min 85° C. to 75°C. (−0.3° C./s) 75° C. 1 min 75° C. to 65° C. (−0.3° C./s) 65° C. 1 min65° C. to 55° C. (−0.3° C./s) 55° C. 1 min 55° C. to 45° C. (−0.3° C./s)45° C. 1 min 45° C. to 35° C. (−0.3° C./s) 35° C. 1 min 35° C. to 25° C.(−0.3° C./s) 25° C. 1 min  4° C. Hold

A T7E1 Master Mix of water, NEB2 buffer, and T7E1 enzyme is prepared.Multiply for each reaction, plus extra. Table 8 shows the components ofthe T7E1 master mix.

TABLE 8 Reaction components for T7E1 Master Mix. 1X reaction Water 7.5μL NEB 2   2 μL buffer T7E1 0.5 μL enzyme PCR  10 μL product Total  20μL

To each reaction, in a 200 μL, strip cap tube, the following reagentsare added: T7E1 master mix (10 μL), and PCR sample (10 μL). The reactionis incubated at 37° C. for 25 minutes.

Loading buffer is added to the sample and the entire sample is run on a3% gel at 120 V for 20 minutes. The gel may be run longer if moreresolution is required. Image and save the gel image.

The image is quantified to determine the amount of cleavage of thetarget nucleic acid.

Example 32 Cellular Testing of Nucleic Acid-Targeting Nucleic AcidVariants

This example shows that nucleic acid-targeting nucleic acid variantsdepicted in FIGS. 22, 24 and 25 and Example 26 were tested in acell-based assay to determine if the in vitro functionality determinedin Example 26 matched in vivo functionality. HEK293 cells were grown to60-70% confluence in 10 cm dishes. Cells were removed by trypsinization,counted using a hemocytometer and then separated in to aliquots of 7×10⁴cells for each well to be transfected. For each well, 250 ng pCB045plasmid expressing mammalian codon-optimized Cas9 was mixed with 30 ngguide RNA and 40 ng copGFP DNA and 0.5u1 Lipofectamine 2000 in a finalvolume of 50u1 DMEM. DNA and lipid were incubated for 15 minutes priorto transfection. Transfections were carried out at the time of plating,by adding the lipid:DNA mix to 450u1 DMEM+10% Fetal Bovine Serumcontaining 7×10⁴ cells. The transfection cell mix was added to 96 welltissue culture plates coated with Rat Tail Collagen I. Cells wereincubated at 37 C in an incubator with 5% CO2 for 40 hours.

Media was removed from each of the wells and cells were lysed usingQuickextract solution (Epicentre) according to manufacturer'sinstructions. DNA harvested from QuickExtract lysis was diluted 1:10 andused as a template in PCR reactions for T7E1 assays as described inExample 30.

FIG. 36 indicates that all variants except v8 and v9 were able to cleavetarget nucleic acid. Nucleic acid-targeting nucleic acid variant v8 wasalso substantially inactive in in vitro assays as depicted in FIG. 23B.Nucleic acid-targeting nucleic acid variant v9 was very weakly active inthe in vitro assay depicted in FIG. 23B.

Example 33 Determining a Cell Fate with a Tagged Cell

This example describes how to track a cell developing from a celllineage. A hematopoietic stem cell (e.g., a hemocytoblast) is contactedwith a site-directed polypeptide, a nucleic acid-targeting nucleic acid,and a donor polynucleotide. The site-directed polypeptide and nucleicacid-targeting nucleic acid form a complex and target a region of thehematopoietic genome for cleavage. Once cleaved, the donorpolynucleotide is inserted into the cleaved site in the hepatopoieticcell's genome. The hematopoietic stem cell is induced to differentiatethrough normal differentiation processes. At different stages ofdifferentiation the sample comprising the differentiated hematopoieticcells can be assayed for the presence of the donor polynucleotide. Inthis way, the differentiation process of a cell can be tracked.

Example 34 Clone Double-Stranded Oligonucleotide Encoding a NucleicAcid-Targeting Nucleic Acid into a Linearized Vector

This example describes how to generate a double-stranded oligonucleotideencoding a portion of nucleic acid-targeting nucleic acid (e.g., aspacer) and insert it into a linearized vector. The linearized vector ora closed supercoiled vector comprises a sequence encoding asite-directed polypeptide (e.g., Cas9), a promoter driving expression ofthe sequence encoding the site-directed polypeptide (e.g., CMVpromoter), a sequence encoding a linker (e.g., 2A), a sequence encodinga marker (e.g., CD4 or OFP), a sequence encoding portion of a nucleicacid-targeting nucleic acid, a promoter driving expression of thesequence encoding a portion of the nucleic acid-targeting nucleic acid,and a sequence encoding a selectable marker (e.g., ampicillin), or anycombination thereof.

Equal amounts of two single-stranded oligonucleotides are annealedtogether (e.g., 50 micromolar). The two single-stranded oligonucleotidescan hybridize together. At least one of the two single-strandedoligonucleotides is complementary to a target nucleic acid (e.g., a10-30 nucleotide region adjacent to a protospacer adjacent motif in atarget). At least one of the two single-stranded nucleotides comprises a3′ overhang sequence comprising the sequence 5′-GTTT-3′. At least one ofthe two single-stranded oligonucleotides comprises a 3′ overhangcomprising the sequence 5′-CGGTG-3′. In some instances, one of the twosingle-stranded oligonucleotides comprises a 5′-GTTT-3′ overhang and theother of the two single-stranded oligonucleotides comprises a5′-CGGTG-3′. Annealing is performed in an annealing buffer comprising atleast 10 mM tris HCl pH 8.0, 1 mM EDTA, pH 8.0, and 100 mM NaCl.Annealing is performed by heating the oligonucleotide mixture at 95 Cfor 3-5 minutes, removing the oligonucleotide mixture from the heatingsource, and allowing the mixture to cool to room temperature for 5-10minutes. The double-stranded oligonucleotide mixture is centrifugedgently. After annealing the mixture may be stored at 4 C or −20 C. Themixture, now of double-stranded oligonucleotides, is diluted to preparetwo stock solutions of 500 nanomolar and 5 nanomolar. The stocksolutions are prepared by diluting the oligonucleotide mixture in water.

The double-stranded oligonucleotide (dsOligonucleotide) is ligated intoa linearized vector. The linearized vector comprises a sequence encodinga site-directed polypeptide (e.g., Cas9), a marker protein (e.g., orangefluorescent protein), and/or a sequence encoding a nucleicacid-targeting nucleic acid, wherein the linearized vector is linearizedat a region of the sequence encoding the nucleic acid-targeting nucleicacid, such that the sticky ends generated match the overhang ends of thedsOligonucleotide. The ligation reaction can comprise 1× ligation buffer(e.g., 50 mM Tris-HCl pH 7.6, 5 mM MgCl₂, 1 mM ATP, 1 mM DTT, and/or 5%PEG 8000), 30 nanogram linearized vector, 5 nM dsOligonucleotide, andDNA ligase (e.g., 4 microliters 5× ligation buffer, 2 microliterslinearized vector at 15 nanogram/microliter, 2 microliters 5 nanomolardsOligonucleotide, 11 microliters water, 1 microliter T4 DNA ligase).The reaction is mixed. The reaction is incubated at room temperature for10 minutes-2 hours. The reaction is placed on ice and transformed intocompetent cells.

Transformation into competent cells comprises transforming intochemically competent TOP10 E. coli cells. Competent cells are thawed onice. 3 microliters of the reaction mixture is added to the competentcells and mixed gently. The cells are incubated on ice for 10-30minutes. The cells are heat-shocked for 30 seconds at 42 C. The cellsare transferred to ice for 2 minutes. 250 microliters of medium (SOC orLB) is added to teh cells. The cells are shaked at 200 rpm for 1 hour at37 C. The cells are then spread on an agar plate comprising 100micrograms/milliliter ampicillin and stored overnight at 37 C.

The transformants are analyzed. For example, the transformants areanalyzed to determine the identity of the dsOligonucleotide ligated intothe vector, and/or confirm the ligation is not a false positive. Toanalyze transformants, colonies are picked and cultured overnight in LBmedium comprising 100 micrograms/milliliter ampicillin at 37 C. Theplasmid comprising the site-directed polypeptide and dsOligonucleotideis isolated (e.g., by miniprep kit). A sequencing reaction is performedon the isolated plasmid. The sequencing reaction utilizes a sequencingprimer that is designed to sequence the dsOligonucleotide (e.g., thesequencing primer is a U6 sequencing primer that binds to the U6promoter which is located just upstream of the sequence encoding thedsOligonucleotide.

Once a desired dsOligonucleotide insertion is identified, the plasmidcan be stored at −20 C or in a glycerol stock at −80 C. To make aglycerol stock, the original colony comprising the desired plasmid isstreaked on an agar plate comprising 100 micrograms/milliliterampicillin and incubated overnight at 37 C. A single colony is isolatedgrown in LB comprising 100 micrograms/milliliter ampicillin until theculture reaches stationary phase. The culture is mixed with glycerol andflash frozen in liquid nitrogen (e.g., 0.85 mL culture is mixed with0.15 mL glycerol).

The purified plasmid comprising the desired dsOligonucleotide isinserted into a cell line (e.g., mammalian cell line, HeLa) bytransfection. To transfect the plasmid, the plasmid is purified at highconcentrations using for example, a maxi prep kit. The plasmid istransfected with lipid-based buffer (e.g., Lipofectamine 2000) intocells which are plated at 70% confluency. 3 micrograms of the vector istransfected into the cells.

Example 35 Nanoparticle Delivery of an Engineered Nucleic Acid-TargetingNucleic Acid

A nanoparticle encapsulating a nucleic acid encoding an engineerednucleic acid-targeting nucleic acid and a site-directed polypeptide willbe prepared. Nanoparticles will be prepared by mixing DOPE, Chol,DSPE-PEG and C₁₆mPEG-Ceramide at a molar ratio of 18:60:20:1:1 in 10 mLof 90% ethanol (total lipid 30 μmole). The nucleic acid will bedissolved in 10 mL of 20 mM Tris buffer (pH 7.4-7.6). After being heatedto 37° C., the two solutions will be mixed together through a duelsyringe pump and the mixed solution will be subsequently diluted with 20mL of 20 mM Tris buffer (300 mM NaCl, pH 7.4-7.6). The mixture will beincubated at 37° C. for 30 minutes and dialyzed in 10 mM PBS buffer (138mM NaCl, 2.7 mM KCl, pH 7.4). Stable particles will be obtained afterthe removal of ethanol from the mixture by dialysis. The nanoparticlesolution will be concentrated by centrifugation at 3,000 rpm and atemperature of 4° C. The concentrated suspension will be collected aftera given time and will be sterilized by filtration through a 0.22 μmsyringe filter (Millex-GV, Millipore, USA). A homogeneous suspension ofthe nanoparticles comprising the nucleic acid encoding the engineerednucleic acid-targeting nucleic acid and the site-directed polypeptidewill be obtained.

The nanoparticles will be contacted to a cell. The nanoparticle willenter the cell. Inside the cell, the nanoparticle will release thenucleic acid encoding the engineered nucleic acid-targeting nucleic acidand the site-directed polypeptide. The nucleic acid will be transcribedand/or translated to produce an engineered nucleic acid-targetingnucleic acid that binds to a site-directed polypeptide protein, therebyforming a complex. The complex will target a target nucleic acid thathybridizes with the engineered nucleic acid-targeting nucleic acid. Thecomplex will cleave the target nucleic acid.

In some instances, the nanoparticle will further comprise a nucleic acidencoding a donor polynucleotide. When the target nucleic acid is cleavedby the site-directed polypeptide, the donor polynucleotide will beinserted into the site of the cleaved target nucleic acid.

1-26. (canceled)
 27. An engineered nucleic acid targeting nucleic acid,comprising: an engineered CRISPR RNA (crRNA), an engineeredtrans-activating CRISPR RNA (tracrRNA), or a combination thereof,wherein (i) the engineered nucleic acid targeting nucleic acid canassociate with a Cas9 protein to form a nucleic acid targeting nucleicacid-Cas9 complex capable of binding a target DNA, and (ii) theengineered crRNA, the engineered tracrRNA, or the combination thereof isaltered relative to a corresponding wild-type crRNA, tracrRNA, or both;and the engineered nucleic acid targeting nucleic acid comprises atleast one of the following: a duplex variant, a spacer extension, amid-tracr modification, or a tracr extension.
 28. The engineered nucleicacid targeting nucleic acid of claim 27, wherein the duplex variantcomprises at least one of the following modifications: a modification ofa first stem, a modification of a bulge, a modification of a firstduplex, or an affinity tag.
 29. The engineered nucleic acid targetingnucleic acid of claim 28, wherein the modification of the first stemcomprises a Csy4 binding sequence to which a Csy4 protein binds, a Cas5binding sequence to which a Cas5 protein binds, or a Cas6 bindingsequence to which a Cas6 protein binds.
 30. The engineered nucleic acidtargeting nucleic acid of claim 27, wherein the spacer extensioncomprises at least one of the following: a spacer extension less than 10nucleotides in length, a 5′ cap, a riboswitch sequence, a sequence thatforms a hairpin, a sequence that targets the nucleic acid targetingnucleic acid to a subcellular location, a sequence that provides fortracking, a sequence that provides a protein binding site for a protein,a primer binding site, or an affinity tag.
 31. The engineered nucleicacid targeting nucleic acid of claim 30, wherein the sequence thatprovides the protein binding site for the protein comprises a Csy4binding sequence to which a Csy4 protein binds, a Cas5 binding sequenceto which a Cas5 protein binds, or a Cas6 binding sequence to which aCas6 protein binds.
 32. The engineered nucleic acid targeting nucleicacid of claim 27, wherein the mid-tracr modification comprises at leastone of the following modifications: a modification of a P domain, amodification of a second stem, a modification of a third stem, an addednucleic acid binding protein binding site, or an affinity tag.
 33. Theengineered nucleic acid targeting nucleic acid of claim 32, wherein theadded nucleic acid binding protein binding site comprises a Csy4 bindingsequence to which a Csy4 protein binds, a Cas5 binding sequence to whicha Cas5 protein binds, or a Cas6 binding sequence to which a Cas6 proteinbinds.
 34. The engineered nucleic acid targeting nucleic acid of claim27, wherein the tracr extension comprises at least one of the following:a 3′ hybridizing extension, an added nucleic acid binding proteinbinding site, or an affinity tag.
 35. The engineered nucleic acidtargeting nucleic acid of claim 34, wherein the added nucleic acidbinding protein binding site comprises a Csy4 binding sequence to whicha Csy4 protein binds, a Cas5 binding sequence to which a Cas5 proteinbinds, or a Cas6 binding sequence to which a Cas6 protein binds.
 36. Theengineered nucleic acid targeting nucleic acid of claim 34, wherein adonor polynucleotide is hybridized to the 3′ hybridizing extension. 37.The engineered nucleic acid targeting nucleic acid of claim 27, whereinthe tracr extension comprises at least one of the following: a tracrextension having a length of from 1 to 400 nucleotides, a 3′polyadenylated tail, a riboswitch sequence, a sequence that forms ahairpin, a sequence that targets the nucleic acid targeting nucleic acidto a subcellular location, a sequence that provides for tracking, or aprimer binding site.
 38. The engineered nucleic acid targeting nucleicacid of claim 29, wherein the Csy4 protein binds to the Csy4 bindingsequence with a Kd ˜50 pM.
 39. The engineered nucleic acid targetingnucleic acid of claim 27, wherein the engineered nucleic acid targetingnucleic acid is a double guide nucleic acid structure.
 40. Theengineered nucleic acid targeting nucleic acid of claim 27, wherein theengineered nucleic acid targeting nucleic acid is a single guide nucleicacid structure.
 41. The engineered nucleic acid targeting nucleic acidof claim 27, wherein the engineered nucleic acid targeting nucleic acidcomprises DNA.
 42. The engineered nucleic acid targeting nucleic acid ofclaim 27, wherein the engineered nucleic acid targeting nucleic acidcomprises RNA.
 43. The engineered nucleic acid targeting nucleic acid ofclaim 41, wherein the engineered nucleic acid targeting nucleic acidcomprises RNA.